Lipopolyamines as transfection agents and pharmaceutical uses thereof

ABSTRACT

Lipopolyamines useful for the transfection of nucleic acid and methods of preparation thereof are disclosed. The lipopolyamines are of general formula I, in which R4 comprises at least one C10-C22 aliphatic radical.

This application is a 371 of PCT/FR96/01774 filed Nov. 8, 1996.

The present invention relates to novel compounds similar to the familyof lipopolyamines, to pharmaceutical compositions containing them, totheir applications for the in vivo and/or in vitro transfection ofnucleic acids and to a process for their preparation.

Many genetic diseases are associated with an expression defect and/orabnormal expression, that is to say deficient or excessive expression,of one or more nucleic acids. The main aim of gene therapy is to correctgenetic abnormalities of this type by means of the in vivo or in vitrocellular expression of cloned genes.

Today, several methods are proposed for the intracellular delivery ofthis type of genetic information. One of them in particular is based onthe use of chemical or biochemical vectors. Synthetic vectors have twomain functions, to compact the DNA to be transfected and to promote itscellular binding as well as its passage across the plasma membrane and,if necessary, across the two nuclear membranes.

Considerable progress has been achieved in this mode of transfection,with the development of technology based on the use of a cationic lipid.It has thus been demonstrated that a positively charged cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),interferes spontaneously, in the form of liposomes or small vesicles,with DNA, which is negatively charged, to form lipid-DNA complexes,which are capable of fusing with cell membranes, and thus allows theintracellular delivery of DNA. However, although this molecule iseffective in terms of transfection, it has the drawback of beingnon-biodegradable and of having a toxic nature with regard to cells.

Since DOTMA, other cationic lipids have been developed along the linesof this structural model: lipophilic group combined with an amino groupvia a so-called “spacer” arm. Among these, mention may be made moreparticularly of those comprising, as lipophilic group, two fatty acidsor a cholesterol derivative, and additionally containing, if necessary,as amino group, a quaternary ammonium group. DOTAP, DOBT and ChOTB maybe mentioned in particular as representatives of this category ofcationic lipids. Other compounds, for instance DOSC and ChOSC, arecharacterized by the presence of a choline group in place of thequaternary ammonium group. In general, however, the transfectingactivity of these compounds remains fairly low.

Another category of cationic lipids, lipopolyamines, has also beendescribed. For the purposes of the present invention, the termlipopolyamine denotes an amphiphilic molecule comprising at least onehydrophilic polyamine region combined, via a so-called spacer region,with a lipophilic region. The polyamine region of lipopolyamines, whichare cationically charged, is capable of combining reversibly withnucleic acid, which is negatively charged. This interaction compacts thenucleic acid greatly. The lipophilic region renders this ionicinteraction insensitive to the external medium, by coating thenucleolipid particle formed with a lipid film. In compounds of thistype, the cationic group may be represented by the L-5-carboxyspermineradical which contains four ammonium groups, two primary and twosecondary. DOGS and DPPES are in particular among the compounds of thistype. These lipopolyamines are most particularly effective fortransfecting primary endocrine cells.

In point of fact, an ideal synthetic transfecting agent should display ahigh level of transfection, and should do so for a broad spectrum ofcells, should have no toxicity or, failing that, a very minimal toxicityat the doses used, and, lastly, should be biodegradable so as to be ridof any side-effects on the cells treated.

The object of the present invention is, precisely, to propose novellipopolyamines, which are original on the basis of their polyaminefraction, and which can be used effectively in the in vitro and/or invivo transfection of cells and in particular for the vectorization ofnucleic acids.

A first subject of the present invention is lipopolyamines, in D, L orD,L form and their salts, characterized in that they are represented bythe general formula I

in which:

R1, R2 and R3 represent, independently of each other, a hydrogen atom ora group —(CH₂) q—NRR′ with

q able to range between 1, 2, 3, 4, 5 and 6, and doing so independentlybetween the various groups R1, R2 and R3 and

R and R′ representing, independently of each other, a hydrogen atom or agroup —(CH₂)q′—NH₂, q being able to range between 1, 2, 3, 4, 5 and 6,and doing so is independently between the various groups R and R′,

m, n and p represent, independently of each other, an integer which mayvary between 0 and 6 with, when n is greater than 1, m able to takedifferent values and R3 able to take different meanings within thegeneral formula I, and

R4 represents a group of general formula II

in which:

R6 and R7 represent, independently of each other, a hydrogen atom or asaturated or unsaturated C10 to C22 aliphatic radical with at least oneof the two groups being other than hydrogen,

u is an integer chosen between 0 and 10 with, when u is an integergreater than 1, R5, X, Y and r able to have different meanings withinthe different units [X—(CHR5)r—Y]

X represents an oxygen or sulphur atom or an amine group which may ormay not be monoalkylated,

Y represents a carbonyl group or a methylene group R5 represents ahydrogen atom or a side chain of a natural amino acid, which issubstituted if necessary, and

r represents an integer ranging between 1 and 10 with, when r is equalto 1, R5 representing a side chain of a natural amino acid and, when ris greater than 1, R5 representing a hydrogen atom.

For the purposes of the invention, the expression side chain of anatural amino acid is understood in particular to denote chainscontaining amidinium units such as, for example, the side chain ofarginine. As mentioned above, this chain may be substituted withsaturated or unsaturated, linear, branched or cyclic C1 to C24 aliphaticgroups such as, for example, cholesteryl, arachidonyl or retinoylradicals and mono- or polyaromatic groups such as, for example,benzyloxycarbonyl derivatives, benzyl ester derivatives and substitutedor unsubstituted rhodaminyl derivatives.

These novel products of general formula (I) may be in the form ofnon-toxic and pharmaceutically acceptable salts. These non-toxic saltscomprise salts with inorganic acids (hydrochloric acid, sulphuric acid,hydrobromic acid, phosphoric acid and nitric acid) or with organic acids(acetic acid, propionic acid, succinic acid, maleic acid, hydroxymaleicacid, benzoic acid, fumaric acid, methanesulphonic acid and oxalic acid)or with inorganic bases (sodium hydroxide, potassium hydroxide, lithiumhydroxide and lime) or organic bases (tertiary amines such astriethylamine, piperidine and benzylamine).

Representatives of the compounds according to the invention which may bementioned more particularly are the compounds of the following generalsub-formulae:

in which R4, R6 and R7 have the above definitions. Preferably, R4represents therein an NR6R7 group with R6 and R7 appearing insubformulae III to XII as an identical group chosen from (CH₂)₁₇CH₃,(CH₂)₁₁CH₃, (CH₂)₁₃CH₃ or (CH₂)₁₂CH₃.

In a particularly advantageous embodiment, the compounds claimed alsocomprise a targeting element which makes it possible to direct thetransfer of the nucleic acid with which they are combined. Thistargeting element is preferably incorporated, on the compound of generalformula I, in the amino acid side chain featured by the substituent R5.More preferably, the targeting element is attached, covalently ornon-covalently, to the compound according to the invention.

This element may be an extracellular targeting element which makes itpossible to direct the transfer of the nucleic acid towards certaindesired cell types or certain desired tissues (tumour cells, livercells, haematopoietic cells, etc.). In this respect, it may be a cellreceptor ligand present at the surface of the target cell type such as,for example, a sugar, a folate, a transferrin, an insulin, anasialo-orosomucoid protein or any bioactive molecule recognized byextracellular receptors. It may also be an intracellular targetingelement which makes it possible to direct the transfer of the nucleicacid towards certain preferred cell compartments (mitochondria, nucleus,etc.), such as, for example, a nuclear localization signal sequence(nls) which promotes the accumulation of transfected DNA in the nucleus.

More generally, the targeting elements which an be used within thecontext of the invention include sugars, peptides, oligonucleotides,steroids and lipids. They are preferably sugars and/or peptides such asantibodies or antibody fragments, cell receptor ligands or fragmentsthereof, receptors or receptor fragments, etc. In particular, they maybe ligands for growth factor receptors, for cytokine receptors, for celllectin receptors or for receptors for adhesion proteins such asintegrins. Mention may also be made of the receptor for transferrin, forHDL lipids and for LDL lipids. The targeting element may also be a sugarwhich makes it possible to target lectins such as asialoglycoproteinreceptors, or alternatively an antibody Fab fragment which makes itpossible to target the immunoglobulin Fc fragment receptor.

Similarly, it is possible to envisage the combination of a labellingagent of biotin, rhodamine or folate type with a compound of generalformula I, for example on the amino acid side chain R5. This labellingagent may also be a linear or cyclic peptide or pseudopeptide sequencecontaining the epitope Arg-Gly-Asp for recognition of the primary and/orsecondary receptors of adhesion proteins of the integrin type.

Illustrations of the lipopolyamines claimed, which may be mentioned moreparticularly are the following compounds, which are described in greaterdetail in the examples below:

H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGyN[(CH₂)₁₇—CH₃]₂

H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂CON[(CH₂)₁₇—CH₃]₂

H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COArgN[(CH₂)₁₇—CH₃]₂

H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COArg(Z)₂N[(CH₂)₁₇—CH₃]₂

H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLys(rhodamine)N[(CH₂)₁₇—CH₃]₂

H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLys(biotinyl)N[(CH₂)₁₇—CH₃]₂

{(H₂N(CH₂)₃}₂N(CH₂)₄N{(CH₂)₃NH₂}(CH₂)₃NHCH₂COGlyN[(CH₂)₁₇—(CH₃]₂

{H₂N(CH₂)₃}₂N(CH₂)₄N{(CH₂)₃NH₂}(CH₂)₃NHCH₂CON[(CH₂)₁₇—CH₃]₂

{H₂N(CH₂)₂}₂N(CH₂)₂NHCH₂COGlyN[(CH₂)₁₇—CH₃]₂

{₂N(CH₂)₂}₂N(CH₂)₂NHCH₂CON[(CH₂)₁₇—CH₃]₂

NH₂(CH₂)₃NH(CH₂)₄N[(CH₂)₃NH₂]CH₂COGlyN[(CH₂)₁₇CH₃]₂

NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLysN[(CH₂)₁₇CH₃]₂

NH₂ (CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLYS[Cl—Z]N[(CH₂)₁₇CH₃]₂

NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLys[CHO]N[(CH₂)₁₇CH₃]₂

NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLys[Cholesteryl]N[(CH₂)₁₇CH₃]₂

NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLys[Arachidonyl]N[(CH₂)₁₇CH₃]₂

NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGluN[(CH₂)₁₇CH₃]₂

NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlu[N(CH₃)₂]N[(CH₂)₁₇CH₃]₂

NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlu[O—Bz]N[(CH₂)₁₇CH₃]₂

NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlu[Galactosamide]N[(CH₂)₁₇CH₃]₂

NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlu[Glucosamide]N[(CH₂)₁₇CH₃]₂

NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlu[Mannosamide]N[(CH₂)₁₇CH₃]₂

NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH(CH₂)₃CON[(CH₂)₁₇CH₃]₂

NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂CONH(CH₂)₅CON[(CH₂)₁₇CH₃]₂

NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlyN[(CH₂)₁₁CH₃]₂

NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlyN[(CH₂)₁₂CH₃]₂ and

NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlyN[(CH₂)₁₃CH₃]₂.

Compounds which are particularly representative of the present inventionand which may be mentioned more particularly [lacuna] whose generalformula is represented below.

An original solid-phase methodology for preparing asyzmmetricfunctionalized polyamines from which the lipopolyamines according to theinvention are derived has also been developed within the Context of thepresent invention.

Access to asymmetric functionalized polyamines is conventionally limitedby the need for selective introduction of modifications into linear orbranched symmetric polyamines. The chemical differences between theprimary and secondary amine groups of a polyamine require greatselectivity in order to carry out reactions such as alkylations,Michael-type additions or acylations. Furthermore, the selectivityimposed by such chemical differences is not compatible with a selectivealkylation in a group of several primary and secondary amines, as isencountered in polyamines. The conventional response to suchrestrictions is to construct asymmetric functionalized polyamines frommonomer blocks bearing, on the one hand, an amine group capable of being“polyaminated” and, on the other hand, the appropriately protectedasymmetric function. This approach thus requires a tiresome multistepsynthetic strategy and in particular independent protection of thefunctional groups.

The method developed within the context of the present invention hasprecisely the aim of getting rid of the drawbacks hitherto encounteredwith this type of synthesis. Its marked advantage is to leadconveniently and rapidly to polyamines which are selectivelyfunctionalized on a single primary amine group by alkylation orreductive alkylation of symmetric polyamines.

The principle of the process claimed is based on the use of a method ofsolid-phase synthesis to promote a bimolecular reaction between thealkylating reagent and the polyamine, thereby avoiding polyalkylation ofthe latter. More precisely, the present invention relates to a processcharacterized in that it uses the coupling of at least one lipidfraction to at least one asymmetric polyamine fraction, the saidpolyamine fraction having been obtained beforehand by a bimolecularreaction between an alkylating agent covalently attached to a solidsupport and a symmetric polyamine. According to this approach, thealkylating reagent is covalently attached to a polymeric support byesterification or amidation. The symmetric polyamine reacts with thealkylating agent in the solid phase by a bimolecular reaction whichleads to the mono-functionalized asymmetric polyamine attached to thesupport. The free amines of the product are usually protected in thesolid phase with protecting groups BOC or Z type and, lastly, theproducts are cleaved from the solid-phase support. These polyamino acidsare, as is convenient, coupled to the lipid fractions to give thedesired transfecting agents. For this coupling, it is possible to usecommon peptide coupling agents such as BOP, Pybop, BopCl and DCC, forexample. The methodology also makes it possible to assemble the entiretransfecting agent on the solid support with the possible introductionof tracer peptides, sugars or fluorescent probes into the molecules. Ofcourse, it turns out to be possible to carry out this type of graftingon the free lipopolyamine.

The feasibility of the method was demonstrated by the synthesis ofseveral linear or branched, asymmetric and functionalized polyaminoacids.

The subject of the present invention is also any therapeutic applicationof lipopolyamines as described above, either directly or inpharmaceutical compositions.

As explained above, the compounds of general formula I prove to be mostparticularly advantageous for the in vitro and in vivo transfection ofnucleic acids. They efficiently compact DNA and are advantageously ofgreatly reduced toxicity.

In order to obtain a maximum effect for the compositions of theinvention, the respective proportions of the compound of general formulaI and of the nucleic acid are preferably determined such that the ratioR of positive charges in the lipopolyamine considered to negativecharges in the said nucleic acid is optimal. Since this optimal ratiovaries in particular according to the mode of use, namely in vivo or invitro, and according to the cell type to be transfected, it is optimizedfor each particular case. This optimization falls within the competenceof a person skilled in the art.

In the pharmaceutical compositions of the present invention, thepolynucleotide may be either a deoxyribonucleic acid or a ribonucleicacid. It may be sequences of natural or artificial origin, and inparticular genomic DNA, cDNA, mRNA, tRNA, rRNA, hybrid sequences orsynthetic or semi-synthetic sequences of modified or unmodifiedoligonucleotides. These nucleic acids may be of human, animal, plant,bacterial, viral etc. origin. They may be obtained by any techniqueknown to those skilled in the art, and in particular by the screening ofbanks, by chemical synthesis or by mixed methods including the chemicalor enzymatic modification of sequences obtained by the screening ofbanks. They may moreover be incorporated into vectors, such as plasmidvectors.

As regards more particularly the deoxyribonucleic acids, they may besingle- or double-stranded, as well as short oligonucleotides or longersequences. These deoxyribonucleic acids may bear therapeutic genes,sequences for regulating transcription or replication, modified orunmodified antisense sequences, regions for binding to other cellcomponents, etc.

For the purposes of the invention, the term therapeutic gene isunderstood in particular to refer to any gene which codes for a proteinproduct having a therapeutic effect. The protein product thus encodedmay be a protein, a peptide, etc. This protein product may be homologouswith respect to the target cell (that is to say a product which isnormally expressed in the target cell when the latter exhibits nopathology). In this case, the expression of a protein makes it possible,for example, to overcome an insufficient expression in the cell or theexpression of a protein which is inactive or weakly active on account ofa modification, or alternatively of overexpressing the said protein. Thetherapeutic gene may thus code for a mutant of a cell protein, havingincreased stability, modified activity, etc. The protein product mayalso be heterologous with respect to the target cell. In this case, anexpressed protein may, for example, make up or provide an activity whichis deficient in the cell, enabling it to combat a pathology or tostimulate an immune response.

Among the therapeutic products, in the sense of the present invention,which may more particularly be mentioned are enzymes, blood derivatives,hormones, lymphokines, interleukins, interferons, TNF, etc. (FR92/03120), growth factors, neurotransmitters or their precursors orsynthetic enzymes, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF,bFGF, NT3, NT5, HARP/pleotrophin, etc., dystrophin or a minidystrophin(FR 9111947), the CFTR protein associated with mucoviscidosis,tumour-suppressant genes: p53, Rb, RapIA, DCC, k-rev, etc. (FR93/04745), genes coding for factors involved in coagulation: factorsVII, VIII, IX, genes involved in DNA repair, suicide genes (thymidinekinase, cytosine deaminase), haemoglobin genes or genes of othertransport proteins, genes corresponding to the proteins involved in themetabolism of lipids, of apolipoprotein type, chosen fromapolipoproteins A-I, A-II, A-IV, B, C-I, C-II, C-III, D, E, F, G, H, Jand apo(a), metabolic enzymes such as, for example, lipoprotein lipase,hepatic lipase, lecithin cholesterol acyltransferase, 7alpha-cholesterol hydroxylase, phosphatitic acid phosphatase, oralternatively lipid transfer proteins such as cholesterol ester transferprotein and phospholipid transfer protein, an EDL binding protein oralternatively a receptor chosen, for example, from LDL receptors,chylomicron-remnant receptors and scavenger receptors, etc.

The therapeutic nucleic acid may also be an antisense sequence or a genewhose expression in the target cell makes it possible to control theexpression of genes or the transcription of cellular mRNA. Suchsequences may, for example, be transcribed in the target cell intocomplementary RNA of cellular mRNA and thus block their translation intoprotein, according to the technique described in patent EP 140,308. Thetherapeutic genes also comprise the sequences coding for ribozymes whichare capable of selectively destroying target RNAs (EP 321,201).

As indicated above, the nucleic acid may also contain one or more genescoding for an antigenic peptide, capable of generating an immuneresponse in humans or animals. In this particular embodiment, theinvention thus makes it possible to produce either vaccines orimmunotherapeutic treatments applied to humans or to animals, inparticular against microorganisms, viruses or cancers. They may inparticular be antigenic peptides specific for Epstein Barr virus, forHIV virus, for hepatitis B virus (EP 185,573), for pseudo-rabies virus,for syncytia-forming virus, for other viruses or alternatively specificfor tumours (EP 259,212).

Preferably, the nucleic acid also comprises sequences which allow theexpression of the therapeutic gene and/or of the gene coding for theantigenic peptide in the desired cell or organ. These may be sequenceswhich are naturally responsible for expression of the gene consideredwhen these sequences are capable of functioning in the infected cell.They may also be sequences of other origin (responsible for theexpression of other proteins, or even synthetic). In particular, theymay be promoter sequences for eukaryotic or viral genes. For example,they may be promoter sequences derived from the genome of the cell whichit is desired to infect. Similarly, they may be promoter sequencesderived from the genome of a virus. In this regard, there may forexample be mentioned the promoters of genes E1A, MLP, CMV, RSV, etc. Inaddition, these expression sequences may be modified by addition ofactivation sequences, regulation sequences, etc. It may also be aninducible or repressible promoter.

Moreover, the nucleic acid may also contain, in particular upstream ofthe therapeutic gene, a signal sequence which directs the therapeuticproduct synthesized into the secretion pathways of the target cell. Thissignal sequence may be the natural signal sequence of the therapeuticproduct, but it may also be any other functional signal sequence, or anartificial signal sequence. The nucleic acid may also contain a signalsequence which directs the therapeutic product synthesized towards aparticular compartment of the cell.

In another embodiment, the present invention relates to compositionscomprising a nucleic acid, a lipopolyamine as claimed and an adjuvantcapable of associating with the lipopolyamine/nucleic acid complex andof improving the transfecting power thereof. The Applicant has indeedshown that the transfecting power of lipopolyamines may, unexpectedly,be increased in the presence of certain adjuvants (lipids, peptides orproteins for example), capable of associating with thelipopolyamine/nucleic acid complex.

In this respect, the compositions of the invention may comprise one ormore neutral lipids as adjuvants. Such compositions are particularlyadvantageous, especially when the ratio R is low. The Applicant hasindeed shown that the addition of a neutral lipid makes it possible toimprove the formation of the nucleolipid particles and, surprisingly, topromote the penetration of the particle into the cell by destabilizingits membrane.

More preferably, the neutral lipids used in the context of the presentinvention are lipids containing 2 fatty chains.

In a particularly advantageous manner, natural or synthetic lipids,which may be zwitterionic or devoid of ionic charge under thephysiological conditions, are used. They may be chosen more particularlyfrom dioleoylphosphatidylethanolamine (DOPE),oleoylpalmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl,-myristoyl phosphatidylethanolamine as well as derivatives thereofN-methylated 1 to 3 times, phosphatidylglycerols, diacylglycerols,glycosyldiacylglycerols, cerebrosides (such as galactocerebrosides inparticular), sphingolipids (such as sphingomyelins in particular) oralternatively asialogangliosides (such as asialoGM1 and GM2 inparticular).

These various lipids may be obtained either by synthesis or byextraction from organs (example: the brain) or from eggs, by standardtechniques well known to those skilled in the art. In particular, theextraction of natural lipids may be performed using organic solvents(see also Lehninger, Biochemistry).

Very recently, the Applicant has demonstrated that it is alsoparticularly advantageous to employ, as adjuvant, a compound which is oris not directly involved in the condensation of the said nucleic acid(WO96/25508).

The presence of such a compound in a transfecting composition based on alipopolyamine makes it possible to reduce the amount of this agentconsiderably, with the ensuing beneficial consequences in terms oftoxicology, without having any negative impact on the transfectingactivity of the said composition. On the contrary, this compositionadvantageously has a higher level of transfection.

The expression “compound involved in the condensation of the nucleicacid” is understood to define a compound which directly or indirectlycompacts the nucleic acid. More precisely, this compound may actdirectly on the nucleic acid to be transfected or may act on anassociated compound which itself is directly involved in thecondensation of this nucleic acid. Preferably, it acts directly on thenucleic acid.

According to a preferred embodiment, this agent acting on thecondensation of the nucleic acids consists, partly or totally, ofpeptide units (KTPKKAKKP)-(SEQ ID No. 1) and/or (ATPAKKAA)-(SEQ ID No.2), it being possible for the number of units to range between 2 and 10.In the structure of the compound according to the invention, these unitsmay be repeated continuously or non-continuously. Thus, they may beseparated by biochemical linkages, for example one or more amino acids,or by chemical bonds. Such an agent may also be partly or totallyderived from a histone, from a nucleoline, from a protamine and/or fromone of the derivatives thereof.

Preferably, the compositions of the invention comprise from 0.01 to 20equivalents of adjuvant per one equivalent of nucleic acids on aweight/weight basis and, more preferably, from 0.5 to 5.

The compositions according to the invention may be formulated for thepurpose of topical, cutaneous, oral, rectal, vaginal, parenteral,intranasal, intravenous, intramuscular, subcutaneous, intraoccular,transdermal, etc. administration. The pharmaceutical compositions of theinvention preferably contain a vehicle which is pharmaceuticallyacceptable for an injectable formulation, in particular for directinjection into the desired organ, or for topical administration (to skinand/or mucous membrane). They may in particular be sterile, isotonicsolutions or dry compositions, in particular freeze-dried compositions,which, by addition depending on the case of sterilized water or ofphysiological saline, allow injectable solutions to be made up. Thedoses of nucleic acid used for the injection and the number ofadministrations may be adapted according to various parameters, and inparticular according to the mode of administration used, the pathologyconcerned, the gene to be expressed, or alternatively the desiredduration of the treatment. As regards more particularly the mode ofadministration, this may be either a direct injection into the tissuesor the circulatory pathways, or a treatment of cells in culture followedby their reimplantation in vivo, by injection or graft.

The present invention thus provides a particularly advantageous methodfor the treatment of diseases, comprising the in vivo or in vitroadministration of a nucleic acid capable of correcting the said disease,in combination with a compound of general formula I under the conditionsdefined above. More particularly, this method may be applied to diseasesresulting from a deficiency of a protein or nucleic acid product and thenucleic acid administered codes for the said protein product or containsthe said nucleic acid product.

The invention covers any use of a lipopolyamine according to theinvention for the in vivo or in vitro transfection of cells.

The present invention will be described more fully using the examplesand figures which follow, which should be considered as beingnon-limiting illustrations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. (1): Measurement of the transfection efficacy after treatment ofNIH 3T3 cells (mouse embryonic cells—fibroblasts) with differentcationic lipids.

FIG. (2): Measurement of the transfection efficacy after treatment ofrabbit SMC cells (primary culture of smooth muscle cells from rabbitaorta) with different cationic lipids.

FIG. (3): Measurement of the transfection efficacy after treatment of3LL cells (Lewis lung carcinoma) with different cationic lipids.

FIG. (4): Measurement of the transfection efficacy after treatment ofNIH 3T3 cells (mouse embryonic cells—fibroblasts) with differentcationic lipids.

FIG. (5): Effect of the DOPE concentration on the transfection efficacyof 3LL cells.

FIG. (6): Transfection of NIH 3T3 cells with variable amounts of DNA anda constant nanomoles lipofectant/μg of DNA ratio.

ABBREVIATIONS AND SYMBOLS

EtOAc: Ethyl acetate

BOC: t-Butoxycarbonyl

BOP: Benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate

DCC: Dicyclohexylcarbodiimide

DCU: Dicyclohexylurea

DMAP: 4-Dimethylaminopyridine

DMF,: Dimethylformamide

DMSO: Dimethyl sulphoxide

DODA: Dioctadecylamine

PE: Petroleum ether

EtOH: Ethanol

Et₃N: Triethylamine

Rf: Coefficient of frontal retention

TFA: Trifluoroacetic acid

THF: Tetrahydrofuran

TMS: Tetramethylsilane

UV: Ultraviolet

SPPS: Solid phase peptide synthesis

HPLC: High pressure liquid chromatography

Z: Benzyloxycarbonyl

ClZ: p-Chlorobenzyloxycarbonyl

A—EQUIPMENT AND METHODS FOR THE CHEMICAL SYNTHESES 1 EQUIPMENT

a) Compounds

The starting polyamines are commercially available, for example:spermidine, spermine, tris(2-aminoethyl)amine, phenylenediamine,diamino-ethane (-propane, -butane, -pentane, -hexane, etc.), or may besynthesized by standard methods, for example by exhaustivecyanoethylation of commercially available amines such as diamino-ethane(-propane, -butane,-pentane, -hexane, etc.) amine, spermidine orspermine, to give branched polyamines.

The alkylating agents are chosen, as a function of the method ofalkylation, as follows: For a standard alkylation: bromoacetic acid,ω-halocarboxylic acids.

For a reductive alkylation: an ω-aldehyde-carboxylic acid, such asglyoxylic acid, succinic semialdehyde, etc., or a keto acid such asacetoacetic acid or pyruvic acid, etc.

The polymers used are resins which are commercially available forsolid-phase peptide synthesis (Merrifield synthesis), for exampleO-chlorotrityl chloride resin and HMP resin, which give products bearingfree acid functions, or a resin of Rink type. The polyamino acids may besynthesized directly on a peptide presynthesized on the solid phase andbearing a bromoalkyl function or an ω-aldehyde acid.

Dioctadecylamine, triethylamine, trifluoroacetic acid, BOP, DMAP andbenzyl chloroformate are commercial products, obtained from Aldrich. TheNaCl and NaHCO₃ solutions are saturated; the KHSO₄ solution is 0.5 M.

b) Physical Measurements

The proton NMR spectra were recorded on Bruker 400 and 600 MHzspectrometers.

The mass spectra were acquired on an API-MS/III machine.

c) Chromatographic Techniques

The HPLC analyses are performed on a Merck-Hitachi machine equipped withan AS-2000A autosampler, an L-6200A intelligent pump and an L-4000UV-visible detector with adjustable wavelength set at 220 nm foranalytical separations and at 235 nm for preparative separations. Thecolumns for the analytical separations are BU-300 aquapore Butyl 7 m,300 A 300×4.6 mm columns from Perkin-Elmer and for the preparativeseparations are Biosil C18 HL 90-10 250×10 mm columns from Biorad. Themobile phases are H₂O (0.1% TFA) and acetonitrile (0.1% TFA). The flowrate for the analytical analyses is adjusted to 1 ml/min, and, for thepreparative analyses, to 4 ml/min.

The thin layer chromatographies (TLC) were carried out on Merck silicagel plates 0.2 mm in thickness.

The column chromatographies were carried out on Merck 60 silica gel ofparticle size 0.063-0.200 mm. They are revealed either with UV (254 nm),with ninhydrin, by spraying (light spray) an ethanolic solution ofninhydrin (40 mg/100 ml EtOH) to reveal the amines or amides by heatingto 150° C., with fluorescamine, by spraying a solution (40 mg/100 mlacetone) to reveal the primary amines, or with iodine, by covering theplate with iodine powder.

The column chromatographies were carried out on Merck 60 silica gel ofparticle size 0.063-0.200 mm.

d) SPPS Technique of Solid-phase Synthesis

The solid-phase synthesis is performed in a handmade SPPS peptidesynthesis manual reactor and the stirrer is a Flask Shaker modelA5-6021. The progress in the coupling of the polyamines to the solidphase and the progress in the protection of the polyamines in the SPPSis monitored by the Kaiser test [Kaiser, E., Colescolt, D. L.,Bossinger, C. D. and Cook, P. I. Anal. Biochem. 34(2), 595 (1970)]. Theresin used in the examples for the SPPS is chlorotrityl chloride resinfrom Novabiochem-Suisse.

2—GENERAL PROCEDURE

a)—Synthesis of Symmetric Polyamines Illustrated by the Preparation of(N,N,N′,N′-tetraaminopropyl)-1,4-diaminobutane:

147 g of 1,4-diaminobutane and 1000 ml of demineralized water are loadedinto a 2-liter three-necked round-bottomed flask. The solution isstirred magnetically. 443 g of acrylonitrile are added over 1 hour via apressure-equalized dropping funnel, the temperature being maintained at38° C. A reflux condenser is then mounted on the flask and the reactionmass is maintained at 80° C. on a water bath for 1 hour. Thefluorescamine test proves to be negative and the excess acrylonitrile isevaporated off under vacuum at 40° C.

Two phases are obtained. The lower organic phase is separated out,washed with 300 ml of water and transferred into a 1000 mlround-bottomed flask. 170 ml of a water/methanol mixture (1:1 v/v) areadded. The resulting mixture is left to crystallize overnight. Thefollowing day, the crystals are filtered off on a 500 ml sinter funnelof porosity 3.

The filter cake on the sinter funnel is washed with methanol (2×170 ml)and ether (2×150 ml). The product is dried on a dish in a desiccatorunder vacuum (26 mm) overnight. 461 g of product are thus obtained (93%yield). The product was analysed by NMR and MS and the analyses are inagreement. The product is hydrogenated without further purification.

30 g of the above polynitrile (0.1 mol) are loaded into a 1-literstainless steel autoclave. A solution of 140 ml of ethanol (95%) and 8 gof NaOH (0.2 mol) is prepared at the same time in a beaker. When thesodium hydroxide has dissolved, this solution is loaded into theautoclave. Nitrogen is passed into the autoclave and 8 ml of Raneynickel on charcoal are loaded in. The autoclave is closed. The initialhydrogenation pressure is 52 atm and it falls to 28.5 atm over 5 hoursat room temperature. The suspension is filtered on paper, the filter iswashed with ethanol (2×25 ml) and the filtrates are concentrated todryness under vacuum. The oil is mixed with 30 ml of water and extractedwith 100 ml of CH₂Cl₂. The organic phase is dried over MgSO₄, filteredand then evaporated under vacuum. A yellowish fluid oil is obtained (27g, 85% yield).

The product was analysed by TLC (single spot), NMR and MS and theanalyses were in agreement. The product is used without furtherpurification.

b)—Method A: Anchoring of an Acidic Function to the Polymeric Support:

Chlorotrityl chloride resin (5 g, 1.2 mmol Cl/g resin) is loaded into anSPPS reactor, 50 ml of CH₂Cl₂ are added and the mixture is stirred for 5min. Bromoacetic acid (1.05 g, 7 mmol) is added, followed by DIEA (0.95ml, 7.5 mmol). The reactor is stirred for two hours at room temperature.The liquid is filtered and the resin is washed with CH₂Cl₂ and iPrOH(10×50 ml) and MeOH (2×50 ml). Lastly, the resin is dried under a streamof nitrogen.

c)—Method B: Reaction of the Polyamines with the Bromoacetyl Resin:

The polyamine (10 molar excess) is dissolved in 50 ml of CH₂Cl₂ andloaded into a reactor containing the product obtained by method A. Thereactor is stirred for 2 h at room temperature. The solvent is filteredand the resin is washed with CH₂Cl₂ and iPrOH (10×50 m); the Kaiser testis positive.

d)—Protection of the Polyamino Acids on the Resin:

Method C:

Di-tert-butyl dicarbonate (48 mmol) and DIEA (50 mmol) are dissolved inCH₂Cl₂ (50 ml) and loaded into a reactor containing the product obtainedaccording to method B. The reactor is stirred overnight. The followingday, the Kaiser test is negative. The solvent is filtered and the resinis washed alternately with CH₂Cl₂ and iPrOH (10×50 ml), MeOH (2×50 ml)and ether (2×50 ml). The resin is dried under a stream of nitrogen. TheKaiser test is still negative.

Method D:

The resin obtained by method B (1.5 g) is loaded into a round-bottomedflask and CH₂Cl₂ (20 ml) is added, followed by DIEA (20 mmol). Themixture is stirred magnetically and benzyl chloroformate (14 mmol) isadded dropwise over 5 min. The pH is maintained at 11 by addition ofDIEA. The following day, the resin is passed into an SPPS reactor,filtered and washed alternately with CH₂Cl₂ and iPrOH (10×20 ml) andether (2×20 ml). The resin is dried under a stream of nitrogen.

e)—Method E: Cleavage of the Protected Polyamino Acids from the Resin:

The resins obtained by methods C and D are loaded into a 250 mlround-bottomed flask equipped with a magnetic stirrer-bar. A solutioncomposed of 50 ml of CH₂Cl₂ and 25 ml of CF₃CH₂OH is added and themixture is stirred for 2 h. The solution is filtered, the resin iswashed with CH₂Cl₂ (2×10 ml) and the organic phases thus obtained arecombined and evaporated under vacuum. The products are then purified byflash chromatography on SiO₂ with CHCl₃/MeOH (9:1) as eluent. Thefractions containing the products are identified by TLC. (For furtherdetails see the examples below).

f)—Method F: Coupling of the Amino Acids with Dilipidylamines:

Boc-amino acid (10 mmol) and C12-C22 dilipidylamine (10 mmol) are loadedinto a 250 ml round-bottomed flask. CHCl₃ (100 ml) is added and themixture is stirred until dissolution is complete. TEA (30 mmol) and BOP(33 mmol) are then added. The pH is maintained at 10 with TEA and thereaction is stirred for 2 h. When the reaction is complete (TLC), thechloroform is evaporated off and the solid is taken up in ethyl acetate(300 ml). The organic phase is washed with KHSO₄ (4×100 ml), NaHCO₃(4×100 ml), and NaCl (4×100 ml). The organic phase is dried over MgSO₄₁,filtered and evaporated under vacuum. The products are analysed by TLC,NMR and MS and are used without further purification. The yields areabout 90%.

g)—Coupling of the Protected Polyamino Acids with Dilipidyl Acid Amidesand Cleavage of the Boc and Z Protecting Groups

Method G

The product obtained by method F (9 mmol) is loaded into around-bottomed flask equipped with a magnetic stirrer-bar and cold (4°C.) TFA (30 ml) is added. The solution is stirred for 1 h. The TFA isevaporated off under vacuum. The product is dissolved by addition of DMF(70 ml). TEA (30 mmol) is added, followed by the protected polyaminoacid obtained by method E (9 mmol). The pH is adjusted to 10 and BOP (33mmol) is added. The solution is stirred for 2 h and monitored by TLC.When the coupling is complete (TLC), KHSO₄ solution is added (700 ml)and the product is extracted with ethyl acetate (3×100 ml). The organicphase is washed with KHSO₄ (3×50 ml), NaHCO₃ (3×50 ml) and NaCl (3×50ml), dried over MgSO₄, filtered and evaporated under vacuum. Theproducts are analysed by NMR, TLC and MS and are deprotected withoutprior purification. TFA (50 ml) is added to the product and the solutionis stirred for 1.5 h, then the TFA is evaporated off. If the productstill contains Z or ClZ groups which are not cleavable with TFA, methodH is followed directly. The final products are purified bysemi-preparative HPLC (see examples).

Method H

The products obtained by method G containing Z or ClZ groups are loadedinto a round-bottomed flask equipped with a magnetic stirrer-bar and aredissolved in 10 ml of MeOH/g of product. Pd/C (10%, 1 g/g of product)and ammonium formate (1 g/g of product) are added at room temperature.The hydrogenation is monitored by HPLC. After 2 h the reaction iscomplete, the mixture is filtered and the filter is washed with 10 ml ofMeOH. Double-distilled water is added and the solution is frozen andfreeze-dried. The final products are purified by preparative HPLC.

h)—Method I for Deprotection of the Boc Protecting Groups

Trifluoroacetic acid (50 ml) is added to the product containing the Bocgroups (1 mmol) in a round-bottomed flask. The solution is stirred for1.5 h and the TFA is evaporated off. The amine is completely deprotectedand ready for use in the couplings without further purification.

B—EQUIPMENT AND METHOD FOR THE BIOLOGICAL STUDY 1. PLASMIDS USED FOR THEIN VITRO TRANSFER OF GENES

The plasmid pCMV-LUC is a construction derived either from the plasmidpGL2-basic vector (Promega) or from the plasmid pGL2-control vector(Promega) by insertion of an Mlu I-Hind III fragment containing thehuman cytomegalovirus (CMV) promoter extracted from the pcDNA3 vectorplasmid (Invitrogen).

2. PROCEDURE FOR PREPARATION OF THE SOLUTIONS USED FOR THE TRANSFECTION

The products described in the invention are dissolved to a concentrationof 20 mM in ethanol or in water, and are then diluted in water, takingcare to ensure that the final ethanolic concentration is less than 10%.

The nucleic acid solutions diluted in physiological saline (0.15M NaCl)are added to the lipofectant solutions in a 1/1 (v/v) ratio. Aftervortex homogenization and incubation for 15 minutes at room temperature,the DNA/lipofectant solutions are distributed, at a final concentrationof 9% (v/v), into wells in which the cells have been washed withprotein-free growth medium (serum) and taken up in growth mediumcontaining or free of serum.

C—EQUIPMENT FOR THE IN VIVO TESTS 1. EQUIPMENT

a) Experimental Models:

6 adult (>8 weeks) female C57/BL mice

tumours of type 3LL (Lewis lung carcinoma) obtained by passing tumourfragments from animal to animal, implanted on the flank under the skin.

b) Plasmids Used:

pXL 2622: this is derived from pGL2 basic (Promega) in which thecytomegalovirus (CMV) promoter extracted from pCDNA3 (Invitrogen) hasbeen inserted upstream of the gene coding for luciferase. This plasmidis obtained by the technique of precipitation with PEG (Ausubel) and isstored in 10 mM Tris 1 mM EDTA pH 8 at 4° C. at a concentration of about10 μg of DNA per μl.

2. PROCEDURES

Solutions injected: the DNA to be transfected is first dissolved in thebuffer, the peptide (KTPKKAKKP)₂ SEQ ID No. 1 is then added and, after20 minutes, a solution of cationic lipids at high concentration (20 or40 mM) is added to the mixture. After addition of all the products, themixture contains, besides the DNA (at a final concentration of 0.5mg/ml), the peptide (0.75 mg/ml) and the cationic lipid, 150 mM NaCl, 5%D-glucose and 5 mM MES pH 6.2. The injection is carried out 20 to 30minutes after the solution has been prepared.

EXAMPLE 1 Synthesis of H₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlyN[—(CH₂)₁₇CH₃]₂(6)

a—Synthesis of{Boc-[3-(Boc-{4-[Boc-(3-Boc-amino-propyl)amino]butyl}amino)propyl]amino}aceticacid (3)

The resin obtained according to method A is reacted with spermineaccording to methods B, C and E. The protected product is purified bychromatography on SiO₂. The yield is 40%.

TLC: R_(f)=0.32 (CHCl₃/MeOH, 9:1); HPLC, R_(t)=4.22 min (H₂O/MeCN: 3 min[40/60], 3-20 min [0/100], 35 min [0/100]; ¹H NMR spectrum (400 MHz,(CD₃)₂SO-d₆ with addition of a few drops of CD₃COOD-d₄, δ in ppm): 1.40(4 s, 36H: C(CH₃)₃); 1.46 (mt, 4H: central CH₂CH₂ of butyl); 1.64 and1.74 (2 mts, 2H each: central CH₂ of the propyls); 2.96 (t, J=7 Hz, 2H:CH₂NCOO); 3.15 (mt, 8H: CH₂NCH₂); 3.23 (t, J=7.5 Hz, 2H: CH₂NCOO); 3.83(s, 2H: OCONCH₂COO); MH⁺: 661.

b—Synthesis of{Z-[3-(Z-{4-[Z-(3-Z-aminopropyl)-amino]butyl}amino)propyl]amino}aceticacid (4)

The resin obtained according to method A is reacted with spermineaccording to methods B, C and D. The protected product is purified bychromatography on SiO₂. The yield is about 20%.

TLC: R_(f)=0.85 (CHCl₃/MeOH, 8:2); HPLC, R_(t)=6.92 min (H₂O/MeCN: 3 min[40/60], 3-20 min [0/100], 35 min [0/100]; ¹H NMR spectrum (400 MHz,(CD₃)₂SO-d₆, at a temperature of 413 K, δ in ppm): 1.49 (mt, 4H: centralCH₂CH₂ of the butyl); 1.74 and 1.81 (2 mts, 2H each: central CH₂ of thepropyls); 3.07 (q, J=7 Hz, 2H: CH₂NCOObenzyl); from 3.15 to 3.30 (mt,8H: CH₂NCH₂); 3.33 (t, J=7.5 Hz, 2H: NCH₂COO); 3.70 (s, 2H: OCONCH₂COO);5.07-5.10-5.12 and 5.13 (4s, 2H each: ArCH₂OCON); 6.65 (unres. mult.,1H: NHCO); from 7.25 to 7.40 (mt, 20H: aromatic H); MH⁺: 797.

c—Boc-Gly-dioctadecylamide. (5)

Boc-Gly is coupled to dioctadecylamine according to method F; 90% yield.

TLC: R_(f)=0.9 (CHCl₃/MeOH, 9:1); MH⁺=679; ¹H NMR spectrum (300 MHz,CDCl₃, δ in ppm): 0.89 (t, J=7 Hz, 6H: CH₃); 1.29 (mt, 60H: central CH₂of the fatty chains); 1.49 (s, 9H: C(CH₃)₃); 1.55 (mt, 4H: 1 CH₂ of eachfatty chain); 3.15 and 3.33 (2t, J=7.5 Hz, 2H each: NCH₂ of the fattychains); 3.95 (d, J=5 Hz, 2H: OCONCH₂CON); 5.57 (unres. mult., 1H:CONH).

d—H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlyN[(CH₂)₁₈]₂ (6)

Products (3) and (5) or (4) and (5) are coupled according to method G.The products are deprotected as described in method G for theBoc-protected product and method H for the Z-protected product. Theproduct is purified by semi-preparative HPLC and the fractions areanalysed by HPLC.

HPLC, R_(t)=15.35 min, (H₂O/MeCN: 3 min [40/60], 3-20 min [0/100], 35min [0/100]; BYK 2 053¹H NMR spectrum (400 MHz, (CD₃)₂SO-d₆ withaddition of a few drops of CD₃COOD-d₄, at a temperature of 300 K, δ inppm): 0.83 (t, J=7 Hz, 6H: CH₃); 1.23 (mt, 60H: central CH₂ of the fattychains); 1.43 and 1.53 (2 mts, 2H each: 1 CH₂ of each fatty chain); 1.63(mt, 4H: central CH₂CH₂ of the butyl); 1.96 (mt, 4H: central CH₂ of thepropyls); 2.93-3.00 and 3.22 (3 mts, 16H in total: NCH₂); 3.83 (s, 2H:NCH₂CON); 4.03 (s, 2H: CONCH₂CON); MH⁺=821.

EXAMPLE 2 Synthesis of H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂CON[(CH₂)₁₈]₂ (7).

Product (3) is coupled with dioctadecylamine according to method F anddeprotected according to method G. The product is purified bysemi-preparative HPLC and the fractions are analysed by HPLC.

HPLC, R_(t)=15.2 min, (H₂O/MeCN: 3 min [40/60], 3-20 min [0/100], 35 min[0/100]; ¹H NMR spectrum (400 MHz, in a mixture of ⅔ CF₃COOD and ⅓CD₃COOD-d₄δ in ppm): 0.78 (t, J=7 Hz, 6H: CH₃); 1.20 (mt, 60H: centralCH₂ of the fatty chains); 1.52 (mt, 4H: 1 CH₂ of each fatty chain); 1.80(mt, 4H: central CH₂CH₂ of the butyl); 2.23 and 2.32 (2 mts, 2H each:central CH₂ of the propyls); from 3.10 to 3.40 (3 mts, 16H in total:NCH₂); 4.15 (s, 2H: NCH₂CON); MH⁺=764.

EXAMPLE 3 Synthesis of H₂N(CH₂)₃NH (CH₂)₄NH—(CH₂)₃NHCH₂COArgN[(CH₂)₁₈]₂(9)

a—Boc-Arg(Z₂)dioctadecylamide (8).

The product is synthesized by coupling of BocArg(Z₂) anddioctadecylamine by method F, in a yield of 91%.

TLC, Rf =0.9 (CHCl₃/MeOH, 9:1); MH⁺=1046.

b—H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COArgN[(CH₂)₁₈]₂ (9)

The product (3) or (4) is coupled with product (8) by method G anddeprotected by method G (Boc) and/or H (Z). The final product ispurified by semi-preparative HPLC and the fractions are analysed byanalytical HPLC.

HPLC, R_(t)=13.83 min, (H₂O/MeCN: 3 min [40/60], 3-20 min [0/100], 35min [0/100]; ¹H NMR spectrum (400 MHz, (CD₃)₂SO—d₆, δ in ppm): 0.90 (t,J=7 Hz, 6H: CH₃); 1.28 (mt, 60H: CH₂ of the fatty chains); from 1.40 to1.80 (mt, 12H: CH₂); 1.93 (mt, 4H: central CH₂ of the propyls); from2.80 to 3.10 (mt, 16H: NCH₂ and NCH₂ of the fatty chains); 3.42 (mt, 2H:CH₂N of the amido); 3.77 (mt, 2H: NCH₂CON); 4.67 (mt, 1H: NCHCON); from6.80 to 7.50 (broad unres. mult., 2H: NH₂); 7.78-7.92-8.80 and 9.03 (mtand 3 unres. mults. respectively, 1H-2H-4H and 1H respectively: CONH—NHand NH₂); MH⁺: 920.

EXAMPLE 4 Synthesis of H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COArg—(Z)₂N[(CH₂)₁₇—CH₃]₂ (10)

Product (3) is coupled to product (8) by method G and the Boc groups arecleaved by the same method. The product is purified by semi-preparativeHPLC and the fractions are analysed by analytical HPLC.

HPLC, R_(t)=17.75 min, (H₂O/MeCN: 3 min [40/60], 3-20 min [0/100], 35min [0/100]; ¹H MMR spectrum (400 MHz, (CD₃)₂SO, δ in ppm): 0.87 (t, J=7Hz, 6H: CH₃); 1.25 (mt, 60H: central CH₂ of the fatty chains); 1.40 and1.57 (2 mts, 2H each: 1 CH₂ of each fatty chain); 1.65 (mt, 8H: centralCH₂CH₂ of the butyls); 1.95 (mt, 4H: central CH₂ of the propyls); from2.85 to 3.05 (mt, 14H in total: NCH₂); 3.23 (t, J=7.5 Hz, 2H: NCH₂);3.75 (s, 2H: NCH₂CON); 3.85 and 3.95 (2 mts, 1H each: CH₂NC); 4.67 (mt,1H: CONCHCON); 5.07 and 5.25 (limiting AB and s respectively, J=13.5 Hz,2H each: NCOOCH₂Ar); from 7.25 to 7.45 (mt, 10H: aromatic H);7.95-8.85-9.00 and 9.20 (4 unres. mults.: exchangeable H); MH⁺: 1188.

EXAMPLE 5 Synthesis of H₂N (CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLys-(rhodamine)N[(CH₂)₁₇—CH₃]₂ (13)

a—Boc-Lys(Z)dioctadecylamide (11)

The product was synthesized by coupling of BocLys(ClZ) withdioctadecylamine by method F, in a yield of 89%.

TLC, Rf=, 92 (CHCl₃/MeOH, 9:1); MH⁺: 918.

b—BocHN(CH₂)₃NBoc(CH₂)₄NBoc(CH₂)₃NBocCH₂COLys-(ClZ)N[(CH₂)₁₇—CH₃]₂ (12)

Product (3) is coupled to product (11) by method G (without deprotectionof the Boc).

¹H NMR spectrum (400 MHz, (CD₃)₂SO-d₆, at a temperature of 423 K, δ inppm): 0.92 (t, J=6.5 Hz, 6H: CH₃); 1.32 (mt, 60H: central CH₂ of thefatty chains); 1.44 (2 s, 36H in total: C(CH₃)₃); from 1.50 to 1.80 (mt,16H: 1 CH₂ of each fatty chain—central CH₂CH₂ of the butyl —CH₂CH₂CH₂and central CH₂ of the propyl); 3.00 (q, J=6.5 Hz, 2H: OCONCH₂); 3.05(g, J=6.5 Hz, 2H: CH₂NCOO); from 3.15 to 3.40 (mt, 14H: NCH₂ of fattychains —CH₂NCH₂ and CH₂NCH₂CH₂N); 3.80 (s, 2H: OCONCH₂CON); 4.75 (mt,1H: CONCHCON); 5.15 (s, 2H: NCOOCH₂Ar); 5.97 and 6.53 (2 mts, 1H each:OCONH and NHCOO); 7.08 (d, J=7.5 Hz, 1H: CONH); from 7.30 to 7.50 (mts,4H: aromatic H).

c—H₂N(HC₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLys(rhodamine)-N[(CH₂)₁₇—CH₃]₂ (13)

The ClZ group on the lysine of product (12) is cleaved by method H andthe product thus obtained is dried under vacuum, taken up in ether andrinsed with NaHCO₃ and NaCl. The ether is dried over MgSO₄ andevaporated under vacuum.

77 mg (60 μmol) of the deprotected product are dissolved in 3 ml ofMeOH, DIEA (64 μl) is added, followed by tetramethylrhodamineisothiocyanate (30 mg, 68 μmol); the solution is stirred for 17 h andthe reaction is monitored by TLC. The following day, the solution isconcentrated to dryness under vacuum. TFA (4 ml) is then added and themixture is left stirring for 1 h. The TFA is evaporated off and thecrude product is purified by semi-preparative HPLC, with a final yieldof 30%.

TLC, Rf=0.05 (MeOH); HPLC(semi-prep.) R_(t)=61.55 min (H₂O/MeCN: 3 min[100/0], 3-45 min [0/100], 45-140 min [0/100]; MH⁺: 1335.

EXAMPLE 6 Synthesis ofH₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLys-(biotinyl)N[(CH₂)₁₇—CH₃]₂ (14)

Product (12) is deprotected by method H (271 mg, 0.21 mmol) and isdissolved in DMF (10 ml). DIEA (0.11 ml) is added, followed by biotin(56.4 mg, 0.23 mmol) and BOP (102 mg, 0.23 mmol); the pH is maintainedat 10 (DIEA) and the end of the reaction is confirmed by thefluorescamine test. The product is recovered as described in method Fand is deprotected, without further purification, with TFA (5 ml) for 1h. The TFA is evaporated off and the product is purified bysemi-preparative HPLC, in a yield of 50%.

HPLC, R_(t)=13.12 min, (H₂O/MeCN: 3 min [40/60], 3-20 min [0/100], 35min [0/100]; MH⁺: 1118.

EXAMPLE 7 Synthesis of{(H₂N(CH₂)₃}₂N(CH₂)₄N{(CH₂)₃NH₂}(CH₂)₃—NHCH₂COGlyN[(CH₂)₁₇—CH₃]₂ (16)

a—{BocNM(CH₂)₃}₂N(CH₂)₄N{(CH₂)₃NHBoc}(CH₂)₃NBocCH₂COOH (15)

Product (1) is anchored to the polymer by method B, protected by methodC and cleaved from the resin by method E. The product is purified onSiO₂ in a yield of 35%.

TLC: R_(f)=0.2 (CHCl₃MeOH, 8:2); ¹H NMR spectrum (400 MHz, (CD₃)₂SO-d₆,with a few drops of CD₃COOD-d₄, at a temperature of 433 K, δ in ppm):1.42 (s, 36H: C(CH₃)₃); 1.56 (mt, 4H: central CH₂CH₂ of the butyl); from1.65 and 1.85 (mt, 8H: central CH₂ of the propyls); 2.76 (mt, 12H:CH₂N(CH₂)₂); 3.06 (t, J=6.5 Hz, 6H: OCONCH₂); 3.29 (mt, 2H: NCH₂); 3.86(s, 2H: OCONCH₂COO); MH⁺=775.

b—{H₂N(CH₂)₃}₂N(CH₂)₄N{(CH₂)₃NH₂}(CH₂)₃—NHCH₂COGlyN[(CH₂)₁₇—CH₃]₂ (16)

Product (15) is coupled with product (5) according to method G. Theproduct is deprotected by method G and is purified by semi-preparativeHPLC, the fractions are analysed by analytical HPLC and arefreeze-dried. 55% yield.

HPLC (semi-prep.): R_(t)=38.72 min (H₂O/MeCN, 10 min [100/0], 10-45 min[0/100], 45-140 min [0/100]; ¹H NMR spectrum (400 MHz, (CD₃)₂SO-d₆, at atemperature of 386 K, δ in ppm): 0.90 (t, J=7 Hz, 6H: CH₃); 1.30 (mt,60H: central CH₂ of the fatty chains); 1.55 (mt, 4H: 1 CH₂ of each fattychain); 1.65 (mt, 4H: central CH₂CH₂ of the butyl); 1.97 (mt, 8H:central CH₂ of the propyls); from 2.80 to 3.05-3.06 and 3.28 (mt and 2 trespectively, J=7.5 Hz, 18H-2H and 4H: NCH₂); 3.80 (s, 2H: NCH₂CON);4.03 (d, J=5.5 Hz, 2H: CONCH₂CON); from 6.00 to 9.00 (broad unres.mult.: NH2 and NH); 8.27 (mt, 1H: CONH). MH⁺: 935.

EXAMPLE 8 Synthesis of{H₂N(CH₂)₃}₂N(CH₂)₄N{(CH₂)₃NH₂}(CH₂)₃—NHCH₂CON[(CH₂)₁₇—CH₃]₂ (17)

This is synthesized as for product (7), using product (15) in place ofproduct (3). The product is purified by semi-preparative HPLC and thefractions are analysed by analytical HPLC and freeze-dried.

HPLC(semi-prep.): R_(t)=38 min (H₂O/MeCN, 10 min [100/0], 10-45 min[0/100], 45-140 min [0/100]; ¹H NMR spectrum (400 MHz, (CD₃)₂SO-d₆, witha few drops of CD₃COOD-d4, δ in ppm): 0.88 (t, J=7 Hz, 6H: CH₃); 1.29(mt, 60H: central CH₂ of the fatty chains); 1.52 (mt, 4H: 1 CH₂ of eachfatty chain); 1.68 (mt, 4H: central CH₂CH₂ of the butyl); from 1.90 to2.10 (mt, 8H: central CH₂ of the propyls); from 2.90 to 2.95 to3.15-3.18 and 3.15 (t, mt and 2 broad t respectively, J=7.5 Hz, 24H intotal: NCH₂); 4.02 (s, 2H: NCH₂CON); MH⁺: 878.

EXAMPLE 9 Synthesis of {H₂N(CH₂)₂}₂N(CH₂)₂NHCH₂COGlyN[(CH₂)₁₇—C₃]₂ (19)

a—{BocNH(CH₂)₂}₂N(CH₂)₂NBocCH₂COOH (18)

This is synthesized as for product (15), using tris(aminoethyl)amine inplace of product (1). 29% yield.

TLC: R_(f)=0.55 (CHCl₃/MeOH, 8:2); ¹H NMR spectrum (400 MHz, (CD₃)₂SO-d₆at a temperature of 393 K, δ in ppm): 1.44 (s, 27H: C(CH₃)₃); 2.58 (t,J=6.5 Hz, 4H: CH₂NCH₂); 2.66 (t, J=7 Hz, 2H: NCH₂); 3.04 (q, J=6.5 Hz,4H: OCONCH₂); 3.28 (t, J=7 Hz, 2H: OCONCH₂); 3.76 (s, 2H: OCONCH₂COO);6.06 (unres. mult., 2H: CONH); MH⁺=505.

b—{H₂N(CH₂)₂}₂N(CH₂)₂NHCH₂COGlyN[(CH₂)₁₇—CH₃]₂) (19)

This is synthesized as for product (17), using product (18) in place ofproduct (15), in a yield of 65%.

HPLC, R_(t)=122 min, (H₂O/MeCN, 10 min [100/0], 10-45 min [0/100],45-140 min [0/100]; ¹H NMR spectrum (400 MHz, (CD₃)₂SO-d₆, with a fewdrops of CD₃COOD-d4, δ in ppm): 0.87 (t, J=7 Hz, 6H: CH₃); from 1.15 to1.35 (mt, 60H: central CH₂ of the fatty chains); 1.45 and 1.55 (2mts,each 2H: 1 CH₂ of each fatty chain); 2.64 (t, J=5.5 Hz, 4H: CH₂NCH₂);2.75 (t, J=6 Hz, 2H: NCH₂); 2.95 (t, J=5.5 Hz, 4H: NCH₂); 3.08 (t, J=6Hz, 2H: NCH₂); 3.25 (mt, 4H: NCH₂ of the fatty chains); 3.88 (s, 2H:NCH₂CON); 4.06 (d, J=5 Hz, 2H: CONCH₂CON); 7.75 (residual broad unres.mult.: NH); 8.68 (residual t, J=5 Hz: CONH); MH⁺: 765.

EXAMPLE 10 Synthesis of {H₂N(CH₂)₂}₂N(CH₂) ₂NHCH₂CON[(CH₂)₁₇—CH₃]₂ (20)

This is synthesized as for product (19), using dioctadecylamine in placeof product (5). 73% yield.

HPLC, R_(t)=100.1 min, (H₂O/MeCN, 10 min [100/0], 10-45 min [0/100],45-140 min [0/100]; MH⁺=708.

EXAMPLE 11 Synthesis ofNH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLysN[(CH₂)₁₇CH₃]₂ (21)

(RPR 127888 A)

Product (12) is deprotected by method H (Cl—Z), followed by method I.The final product is purified by semi-preparative HPLC and the fractionsare analysed by analytical HPLC.

HPLC, Rt=11.76 min, (H₂O/MeCN: 3 min [60/40], 3-20 min [0/100], 35 min[0/100]; MH⁺: 892; ¹H NMR Spectrum (400 MHz, (CD₃)₂SO-d6, at atemperature of 393 K, d in ppm): 0.91 (t, J=7 Hz, 6H: CH₃ of the fattychains); 1.31 (mt, 60H: (central (CH₂)₁₅ of the fatty chains); from 1.35to 1.75 (Mt, 10H: 1 CH₂ of each fatty chain, central (CH₂)₃ of thelysyl); 1.75 (mt, 4H: central (CH₂)₂ of the butyl); 2.00 (mt, 4H: CH₂ ofthe propyls); 2.82-2.98-3.06 and from 3.10 to 3.50 (2 t—mt and 2 unres.mult. respectively, J=7 Hz, 18H in total: NCH₂ of the lysyl —NCH₂ of thebutyl —NCH₂ of the propyls and NCH₂ of the fatty chains); 3.62 (s, 2H:NCH₂CON); 4.73 (q, J=7 Hz, 1H: CONCHCON of the lysyl); 8.18 (d, J=7 Hz,1H: CONH of the lysyl).

EXAMPLE 12 Synthesis ofNH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLys(Cl—Z)N[(CH₂)₁₇CH₃]₂ (22)

(RPR 122759 A)

Product (12) is deprotected by method I. The final product is purifiedby semi-preparative HPLC and the fractions are analysed by analyticalHPLC.

HPLC, Rt=16.79 min, (H₂O/MeCN: 3 min [60/40], 3-20 min [0/100], 35 min[0/100]; MH⁺: 1060; ¹H NMR Spectrum (400 MHz, (CD₃)₂SO-d₆, at atemperature of 373 K, d in ppm): 0.91 (t, J=7 Hz, 6H: CH₃ of the fattychains); 1.31 (mt, 60H: central (CH₂)₁₅ of the fatty chains); from 1.30to 1.75 (mt, 10H: 1 CH₂ of each fatty chain, central (CH₂)₃ of thelysyl); 1.72 (mt, 4H: central (CH₂)₂ of the butyl); 1.95 (mt, 4H: CH₂ ofthe propyls); 2.98-3.06 and from 2.90 to 3.50 (2 mts and unres. mult.respectively, 18H in total: NCH₂ of the lysyl —NCH₂ of the butyl —NCH₂of the propyls and NCH₂ of the fatty chains); 3.59 (s, 2H: NCH₂CON);4.75 (q, J=7 Hz, 1H: CONCHCON of the lysyl); 5.16 (s, 2H: COOCH₂Ar);6.85 (unres. mult., 1H: OCONH); from 7.35 to 7.55 (mt, 5H: aromatic H);8.15 (unres. mult., 1H: CONH of the lysyl).

EXAMPLE 13 Synthesis ofNH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLys(CHO)N[(CH₂)₁₇CH₃]₂ (24)

(RPR 122760 A) (24).

a—NHBoc(CH₂)₃NBoc(CH₂)₄NBoc(CH₂)₃NBocCH₂COLysN-[(CH₂)₁₇CH₃]₂ (23).

Product (12) is deprotected by method H (Cl—Z) in a yield of 65%, and isused without further purification.

HPLC, Rt=20.82 min, (H₂O/MeCN: 3 min [60/40], 3-20 min [0/100], 35 min[0/100].

b—NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLys(CHO)N[(CH₂)₁₇CH₃]₂ (24).

Product (23) is coupled with formic acid by method G. The product ispurified by semi-preparative HPLC and the fractions are analysed byanalytical HPLC.

HPLC, Rt=13.60 min, (H₂O/MeCN: 3 min [60/40], 3-20 min [0/100], 35 min[0/100]; MH⁺: 920; ¹H NMR Spectrum (400 MHz, (CD₃)₂SO-d₆, at atemperature of 383 K, d in ppm): 0.92 (t, J=7 Hz, 6H: CH₃ of the fattychains); 1.31 (mt, 60H: central (CH₂)₁₅ of the fatty chains); from 1.35to 1.70 (mt, 10H: 1 CH₂ of each fatty chain, central (CH₂)₃ of thelysyl); 1.73 (mt, 4H: central (CH₂)₂ of the butyl); 1.98 (mt, 4H: CH₂ ofthe propyls); from 2.85 to 3.50 (mt, 18H: NCH₂ of the lysyl —NCH₂ of thebutyl —NCH₂ of the propyls and NCH₂ of the fatty chains); 3.62 (s, 2H:NCH₂CON); 4.75 (mt, 1H: CONCHCON of the lysyl); 7.60 (unres. mult., 1H:CONH); 8.05 (broad s, 1H: CH of the aldehyde); 8.18 (unres. mult., 1H:CONH of the lysyl).

EXAMPLE 14 Synthesis ofNH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLys[Cholesteryl]N [(CH₂)₁₇CH₃]₂ (25)

(RPR 128142 A)

Product (23) is coupled with cholesteryl chloroformate according tomethod G (without use of the BOP reagent). The product is purified bysemi-preparative HPLC and the fractions are analysed by analytical HPLC.

HPLC, Rt=21.66 min, (H₂O/MeCN: 3 min [60/40], 3-20 min [0/100], 35 min[0/100]; MH⁺: 1304; ¹H NMR Spectrum (600 MHz, (CD₃)₂SO-d₆, d in ppm):0.68 and 0.98 (2 s, 3H each: CH₃ at 18 and CH₃ at 19 of thecholesteryl); 0.86 (mt, 12H: CH₃ of the fatty chains and CH₃ at 26 and27 of the cholesteryl); 0.91 (d, J=7 Hz, 3H: CH₃ at 21 of thecholesteryl); 1.31 (mt, 60H: central (CH₂)₁₅ of the fatty chains); from0.80 to 2.30 (mt, 42H: 1 CH₂ of each fatty chain —CH₂ at 1, 2, 4, 7, 11,12, 15, 16, 22, 23 and 24 of the cholesteryl —CH at 8, 9, 14, 17, 20 and25 of the cholesteryl —central (CH₂)₃ of the lysyl and CH₂ of thepropyls); 1.65 (mt, 4H: central (CH₂)₂ of the butyl); 2.88 and 2.96 (2mts, 14H in total: NCH₂ of the lysyl —NCH₂ of the butyl —NCH₂ of thepropyls); from 3.20 to 3.50 (mt, 4H: NCH₂ of the fatty chains); 3.64 (s,2H: NCH₂CON); 4.23 (mt, 1H: CH at 3 of the cholesteryl); 4.63 (mt, 1H:CONCHCON of the lysyl); 5.30 (mt, 1H: CH at 6 of the cholesteryl); 6.98(mt, 1H: NHCOO); 7.90 (mt, 1H: CONH of the lysyl); 8.60 to 9.10(exchangeable unres. mults.).

EXAMPLE 15 Synthesis of NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLys[Arachidonyl]-N[(CH₂)₁₇CH₃]₂ (26)

(RPR 130605)

Product (23) is coupled with arachidonic acid, under a stream ofnitrogen and sheltered from the light, according to method G. Theproduct is purified by semi-preparative HPLC and the fractions areanalysed by analytical HPLC.

HPLC, Rt=20.67 min, (H₂O/MeCN: 3 min [60/40], 3-20 min [0/100], 35 min[0/100]; MH⁺: 1177; ¹H NMR Spectrum (400 MHz, (CD₃)₂SO-d₆ with additionof a few drops of CD₃COOD-d₄, at a temperature of 393 K, d in ppm): 0.90(t, J=7 Hz, 6H: CH₃ of the fatty chains); 0.91 (t, J=7 Hz, 3H: CH₃ ofthe arachidonyl); 1.31 (mt, 60H: central (CH₂)₁₅ of the fatty chains);from 1.35 to 1.75 (mt, 18H: 1 CH₂ of each fatty chain—central (CH₂)₃ andcentral CH₂ of the arachidonyl and central (CH₂)₃ of the lysyl); 1.75(mt, 4H: central (CH₂)₂ of the butyl); 2.02 (mt, 4H: CH₂ of thepropyls); 2.10 (mt, 6H: COCH₂ and the two ═CCH₂ of the arachidonyl);2.80-2.97-3.06 and from 3.10 to 3.50 (mt—t—mt and 2 unres. mults.respectively, J=7 Hz, 24H in total: ═CCH₂C═of the arachidonyl —NCH₂ ofthe lysyl —NCH₂ of the butyl —NCH₂ of the propyls and NCH₂ of the fattychains); 3.62 (s, 2H: NCH₂CON); 4.73 (dd, J=8 and 5 Hz, 1H: CONCHCON ofthe lysyl); 5.38 (mt, 8H: CH═CH of the arachidonyl).

EXAMPLE 16 Synthesis ofNH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGluN[(CH₂)₁₇CH₃]₂ (28)

(RPR 126097 A)

a—Boc-Glu(O-Bz)-dioctadecylamine (27)

The product is synthesized by coupling of Boc-Glu(OBz) anddioctadecylamine by method F, in a yield of 90%.

TLC Rf=0.88 (CHCl₃/MeOH, 9:1)

b—NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGluN[(CH₂)₁₇CH₃]₂ (28).

Product (3) or (4) is coupled with product (27) by method G, followed bymethod H (Cl—Z deprotection). The final product is purified bysemi-preparative HPLC and the fractions are analysed by analytical HPLC.

HPLC, Rt=14.64 min, (H₂O/MeCN: 3 min [60/40], 3-20 min [0/100], 35 min[0/100]; MH⁺: 893; ¹H NMR Spectrum. (400 MHz, (CD₃)₂SO-d₆, at atemperature of 383 K, d in ppm): 0.90 (t, J=7 Hz, 6H: CH₃ of the fattychains); 1.30 (mt, 60H: central (CH₂)₁₅ of the fatty chains); 1.56(unres. mult., 4H: 1 CH₂ of each fatty chain); from 1.60 to 2.00 (mt,2H: central CH₂ of the glutaryl); 1.73 (mt, 4H: central (CH₂)₂ of thebutyl); 1.98 (mt, 4H: CH₂ of the propyls); 2.32 (t, J=7 Hz, 2H: COCH₂ ofthe glutaryl); 3.00-3.06 and 3.45 (t and 2 mts respectively, J=7 Hz, 16Hin total: NCH₂ of the butyl —NCH₂ of the propyls and NCH₂ of the fattychains); 3.65 (broad s, 2H: NCH₂CON); 4.85 (mt, 1H: CONCHCON of theglutaryl); 8.19 (broad s, 1H: CONH of the glutaryl).

EXAMPLE 17 Synthesis ofNH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlu(O-Bz)N[(CH₂)₁₇CH₃]₂ (29)

(RPR 123027 A)

Product (3) is coupled to product (27) and deprotected by method G(Boc). The final product is purified by semi-preparative HPLC and thefractions are analysed by analytical HPLC.

HPLC, Rt=16.02 min, (H₂O/MeCN: 3 min [60/40], 3-20 min [0/100], 35 min[0/100]; MH⁺: 983; ¹H NMR Spectrum (400 MHz, (CD₃)₂SO-d₆, at atemperature of 413 K, d in ppm): 0.89 (t, J=7 Hz, 6H: CH₃ of the fattychains); 1.30 (mt, 6OH: central (CH₂)₁₅ of the fatty chains); 1.55(unres. mult., 4H: 1 CH₂ of each fatty chain); 1.72 (mt, 4H: central(CH₂)₂ of the butyl); from 1.75 to 2.00 (mt, 2H: central CH₂ of theglutaryl); 1.99 (mt, 4H: CH₂ of the propyls); 2.47 (t, J =7 Hz, 2H:COCH₂ of the glutaryl); 2.95-3.05 and 3.40 (3 mts, 16H in total: NCH₂ ofthe butyl —NCH₂ of the propyls and NCH₂ of the fatty chains); 3.62(broad s, 2H: NCH₂CON); 4.85 (mt, 1H: CONCHCON of the glutaryl); 5.14(limiting AB, J=12 Hz, 2H: CH₂ of the benzyl); 7.35 (mt, 5H: aromatic Hof the benzyl); 8.23 (unres. mult., 1H: CONH of the glutaryl).

EXAMPLE 18 Synthesis ofNH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlu[Galactosamide]—N[(C₂)₁₇CH₃]₂ (31)

(RPR 130596 A)

a—BocNH(CH₂)₃NBoc(CH₂)₄NBoc(CH₂)₃NBocCH₂COGluN—[(CH₂)₁₇CH₃]₂ (30)

Product (3) is coupled to product (27) and the OBz protecting group ofthe side chain is cleaved off by method H (Cl—Z), and the product isused without further purification.

HPLC, Rt=22.84 min, (H₂O/MeCN: 3 min [60/40], 3-20 min [0/100], 35 min[0/100]; MH⁺: 1293.

b—NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlu(Galactosamide)N-[(CH₂)₁₇CH₃]₂ (31)

Product (30) is coupled with D-(+)-galactosamine hydrochloride accordingto method G. The product is purified by semi-preparative HPLC and thefractions are analysed by analytical HPLC.

HPLC, Rt=13.71 min, (H₂O/MeCN: 3 min [60/40], 3-20 min [0/100], 35 min[0/100]; MH⁺: 1054.

EXAMPLE 19 Synthesis ofNH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlu[Galactosamide]-N[(CH₂)₁₇CH₃]₂ (32)

(RPR 130595 A)

Product (30) is coupled with D-(+)-glucosamine hydrochloride accordingto method G. The product is purified by semi-preparative HPLC and thefractions are analysed by analytical HPLC.

HPLC, Rt=12.27 min, (H₂O/MeCN: 3 min [60/40], 3-20 min [0/100], 35 min[0/100]; MH⁺: 1054.

EXAMPLE 20 Synthesis ofNH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlu[Mannosamide]-N[(CH₂)₁₇CH₃]₂ (33)

(RPR 130598 A)

Product (30) is coupled with D-(+)-mannosamine hydrochloride accordingto method G. The product is purified by semi-preparative HPLC and thefractions are analysed by analytical HPLC.

HPLC, Rt=12.98 min, (H₂O/MeCN: 3 min [60/40], 3-20 min [0/100], 35 min[0/100]; MH⁺: 1054.

EXAMPLE 21 Synthesis ofNH₂(CH₂)₃NH(CH₂)₄NH(₂)₃NHCH₂COGlu(N(CH₃)₂N[(CH₂)₁₇CH₃]₂ (34)

(RPR 131111 A)

Product (30) is coupled with dimethylamine according to method G. Theproduct is purified by semi-preparative HPLC and the fractions areanalysed by analytical HPLC.

HPLC, Rt=14.44 min, (H₂O/MeCN: 3 min [60/40], 3-20 min [0/100], 35 min[0/100]; MH⁺: 920; ¹H NMR Spectrum (400 MHz, (CD₃)₂SO-d₆, d in ppm):0.89 (t, J=7 Hz, 6H: CH₃ of the fatty chains); 1.25 (mt, 60H: central(CH₂)₁₅ of the fatty chains); 1.43 and 1.60 (2 mts, 2H each: 1 CH₂ ofeach fatty chain); 1.65 (mt, 4H: central (CH₂)₂ of the butyl); 1.65 andfrom 1.85 to 2.00 (2 mts, 1H each: central CH₂ of the glutaryl); 1.95(mt, 4H: CH₂ of the propyls); 2.32 (limiting AB, 2H: COCH₂ of theglutaryl); 2.80 and 2.92 (2s, 3H each: CON(CH₃)₂); from 2.85 to 3.05(mt, 12H: NCH₂ of the butyl —NCH₂ of the propyls); 3.00-3.22-3.45 and3.58 (4 mts, 1H each: NCH₂ of the fatty chains); 3.78 (AB, J=16 Hz, 2H:NCH₂CON); 4.75 (mt, 1H: CONCHCON of the glutaryl); 8.72 (d, J=7.5 Hz,1H: CONH of the glutaryl); 8.85 and from 8.90 to 9.15 (exchangeableunres. mults.).

EXAMPLE 22 Synthesis ofNH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlyN[(CH₂)₁₂CH₃]; ₂ (35)

(RPR 122767 A)

This is synthesized in the same way as product (6), but usingdidodecylamine in place of dioctadecylamine. The product is purified bysemi-preparative HPLC and the fractions are analysed by HPLC.

HPLC, Rt=9.54 min, (H₂O/MeCN: 3 min [60/40], 3-20 min [0/100], 35 min[0/100]; MH⁺: 653; ¹H NMR Spectrum (400 MHz, (CD₃)₂SO-d₆, at atemperature of 403 K, d in ppm); 0.93 (t, J=7 Hz, 6H: CH₃ of the fattychains); 1.33 (mt, 36H: central (CH₂)₉ of the fatty chains); 1.58 (mt,4H: 1 CH₂ of each fatty chain); 1.75 (mt, 4H: central (CH₂)₂ of thebutyl); 1.95 and 2.00 (2 mts, 2H each: central CH₂ of the propyls); 2.98and 3.00 (2 mts, 12H in total: NCH₂ of the butyl and NCH₂ of thepropyls); 3.30 (t, J=7 Hz, 4H: NCH₂ of the fatty chains); 3.58 (s, 2H:NCH₂CON); 4.05 (s, 2H: CONCH₂CON of the glycyl).

EXAMPLE 23 Synthesis ofNH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlyN[(CH₂)₁₂CH₃]₂ (36)

(RPR 122774 A)

This is synthesized in the same way as product (6), but usingditridecylamine in place of dioctadecylamine. The product is purified bysemi-preparative HPLC and the fractions are analysed by HPLC.

HPLC, Rt=10.64 min, (H₂O/MeCN: 3 min [60/40], 3-20 min [0/100], 35 min[0/100]; MH⁺: 681; ¹H NMR Spectrum (400 MHz, (CD₃)₂SO-d₆, at atemperature of 393 K, d in ppm): 0.91 (t, J=7 Hz, 6H: CH₃ of the fattychains); 1.33 (mt, 40H: central (CH₂)₁₀ of the fatty chains); 1.58 (mts,4H: 1 CH₂ of each fatty chain); 1.75 (mt, 4H: central (CH₂)₂ of thebutyl); 2.00 (mt, 4H: central CH₂ of the propyls); 2.98 and 3.08 (2 t,J=7 Hz, 12H in total: NCH₂ of the butyl and NCH₂ of the propyls); 3.32(t, J=7 Hz, 4H: NCH₂ of the fatty chains); 3.65 (s, 2H: NCH₂CON); 4.06(d, J=4 Hz, 2H: CONCH₂CON of the glycyl); 8.60 (broad s, 1H: CONH of theglycyl).

EXAMPLE 24 Synthesis ofNH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlyN[(CH₂)₁₃CH₃]; ₂ (37)

(RPR 122766 A)

This is synthesized in the same way as product (6), but usingditetradecylamine in place of dioctadecylamine. The product is purifiedby semi-preparative HPLC and the fractions are analysed by HPLC.

HPLC, Rt=9.92 min, (H₂O/MeCN: 3 min [60/40], 3-20 min [0/100], 35 min[0/100]; M⁺: 709; ¹H NMR Spectrum (400 MHz, (CD₃)₂SO-d₆, at atemperature of 393 K, d in ppm): 0.90 (t, J=7 Hz, 6H: CH₃ of the fattychains); 1.31 (mt, 44H: central (CH₂)₁₁ of the fatty chains); 1.58 (mt,4H: 1 CH₂ of each fatty chain); 1.76 (mt, 4H: central (CH₂)₂ of thebutyl); 2.00 (mt, 4H: central CH₂ of the propyls); 2.98 and 3.08 (mt andt respectively, J=7 Hz, 12H in total: NCH₂ of the butyl and NCH₂ of thepropyls); 3.30 (t, J=7 Hz, 4H: NCH₂ of the fatty chains); 3.65 (s, 2H:NCH₂CON); 4.06 (d, J=4 Hz, 2H: CONCH₂CON of the glycyl); 8.10 (unres.mult., 1H: CONH of the glycyl).

EXAMPLE 25 Synthesis ofNH₂(CH₂)₃NH(CH₂)₄N[(CH₂)₃NH₂]CH₂COGlyN[(CH₂)₁₇CH₃]₂ (39)

(RPR 126096 A)

a—Synthesis of BocNH(CH₂)₃NBoc(CH₂) ₄N[(CH₂)₃NHBoc]CH₂CO₂H (38)

During the synthesis of product (3), by-product (38) is recovered duringthe purification on Sio₂.

The yield is 8%. TLC Rf=0.32 (CHCl₃/MeOH, 9:1); MH⁺: 561; ¹H NMRSpectrum (400 MHz, (CD₃)₂SO-d₆, d in ppm): from 1.30 to 1.60 (mt, 4H:central (CH₂)₂ of the butyl); 1.40 (s, 27H: C(CH₃)₃); 1.56 (mt, 4H: CH₂of the propyls); 2.68 and 3.11 (broad t and t respectively, J=7 Hz, 4Heach: NCH₂ of the butyl and NCH₂ of the propyls); 2.90 and 2.96 (2 q,J=7 Hz, 2H each: BocNHCH₂ of the propyls); 3.18 (s, 2H: NCH₂COO).

b—NH₂(CH₂)₃NH(CH₂)₄N[(CH₂)₃NH₂]CH₂COGlyN[(CH₂)₁₇CH₃]₂ (39)

Products (38) and (5) are coupled according to method G. The product ispurified by semi-preparative HPLC and the fractions are analysed byHPLC.

HPLC, Rt=13.60 min, (H₂O/MeCN: 3 min [60/40], 3-20 min [0/100], 35 min[0/100]; MH⁺: 821; ¹H NMR Spectrum (400 MHz, (CD₃)₂SO-d₆ with additionof a few drops of CD₃COOD-d₄, d in ppm): 0.87 (t, J=7 Hz, 6H: CH₃ of thefatty chains); 1.28 (mt, 60H: central (CH₂)₁₅ of the fatty chains); 1.46and 1.54 (2 mts, 2H each: 1 CH₂ of each fatty chain); 1.63 (mt, 4H:central (CH₂)₂ of the butyl); 1.91 (mt, 4H: CH₂ of the propyls); from2.85 to 3.15 (mt, 12H: NCH₂ of the butyl and NCH₂ of the propyls); 3.24(mt, 4H: NCH₂ of the fatty chains); 3.76 (unres. mult., 2H: NCH₂CON);4.05 (broad s, 2H: CONCH₂CON of the glycyl).

EXAMPLE 26 Synthesis ofNH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH(CH₂)₃CON[(CH₂)₁₇CH₃]₂ (41)

(RPR 122786 A)

a—BocNH(CH₂)₃NBoc(CH₂)₄NBoc(CH₂)₃NBoc(CH₂)₃NBoc (CH₂)₃CO₂H(40)

Product (40) is synthesized using reductive alkylation on spermine inthe presence of NaCNBH₃ and succinic semialdehyde in solution.

1.8 g of spermine, 60 ml of methanol and 0.138 g of NaCNBH₃ are loadedinto a 200 ml round-bottomed flask. The solution is placed undervigorous magnetic stirring. A solution of 5.5 ml of succinicsemialdehyde (15%) in 30 ml of methanol is run in, via apressure-equalized dropping funnel, over 100 minutes. Stirring iscontinued for 100 minutes. The amines are protected with the Boc groupas follows: 2.8 ml of TEA are run into the medium, followed by 8.8 g ofdi-tert-butyl dicarbonate dissolved in 30 ml of methanol. Stirring iscontinued overnight. The medium is concentrated under vacuum, theproduct is taken up in ethyl acetate and extracted with three 50 mlfractions of NaHCO₃, and the aqueous phases are combined and rinsed withether (3×100 ml). The pH of the aqueous phase is lowered to 3 withKHSO₄, turbidity is observed due to the precipitation of product (41),and the mixture is extracted with ethyl acetate (3×100 ml). The organicphase is dried over MgSO₄ and evaporated under vacuum. The product isused without further purification.

b—NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH(CH₂)₃CON[(CH₂)₁₇CH₃]₂ (41)

Product (40) and dioctadecylamine are coupled according to method G. Theproduct is purified by semi-preparative HPLC and the fractions areanalysed by HPLC.

HPLC, Rt=15.04 min (H₂O/MeCN: 3 min [60/40], 3-20 min [0/100], 35 min[0/100]; MH⁺: 792; ¹H NMR Spectrum (400 MHz, (CD₃)₂SO-d₆, at atemperature of 383 K, d in ppm): 0.85 (t, J=7 Hz, 6H: CH₃ of the fattychains); 1.22 (mt, 60H: central (CH₂)₁₅ of the fatty chains); 1.48(unres. mult., 4H: 1 CH₂ of each fatty chain); 1.72 (mt, 4H: central(CH₂)₂ of the butyl); 1.88 (mt, 2H: central CH₂ of the aminopentanoyl);1.99 (mt, 4H: CH₂ of the propyls); 2.42 (t, J=7 Hz, 2H: COCH₂ of theaminopentanoyl); 2.96-3.03 and 3.22 (3 mts, 18H in total: NCH₂ of theaminopentanoyl —NCH₂ of the butyl —NCH₂ of the propyls and NCH₂ of thefatty chains).

EXAMPLE 27 Synthesis ofNH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂CONH(CH₂)₅CON[(CH₂)₁₇CH₃]₂ (42)

(RPR 128506 A)

This is synthesized in the same way as product (6), but usingBoc-6-aminocaproic acid in place of BocGly. The product is purified bysemi-preparative HPLC and the fractions are analysed by HPLC.

HPLC, Rt=13.94 min (H₂O/MeCN: 3 min [60/40], 3-20 min [0/100], 35 min[0/100]; MH⁺: 877; ¹H NMR Spectrum (300 MHz, (CD₃)₂SO-d₆, d in ppm):0.87 (t, J=7 Hz, 6H: CH₃ of the fatty chains); 1.28 (mt, 60H: central(CH₂)₁₅ of the fatty chains); 1.48 (mt, 10H: 1 CH₂ of each fatty chainand central (CH₂)₃ of the aminohexanoyl); 1.65 (mt, 4H: central (CH₂)₂of the butyl); 1.95 (mt, 4H: CH₂ of the propyls); 2.27 (t, J=7 Hz, 2H:COCH₂ of the aminohexanoyl); from 2.85 to 3.30 (mts, 18H: NCH₂ of theaminohexanoyl —NCH₂ of the butyl —NCH₂ of the propyls and NCH₂ of thefatty chains); 3.70 (broad s, 2H: NCH₂CON); from 7.90 to 9.10(exchangeable unres. mults.).

EXAMPLE 28 Synthesis ofNH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlu(11-amide-undecanyl,hepta,O acetyllactose) N[(CH₂)₁₇CH₃]₂ (43)

(RPR 130765 A)

Product (30) is coupled with 11-aminoundecanylhepta-O-acetyllactoseaccording to method G. The product is purified by semi-preparative HPLCand the fractions are analysed by analytical HPLC.

HPLC, Rt=15.91 min (H₂O/MeCN: 3 min [60/40], 3-20 min [0/100], 35 min[0/100]; MH⁺: 1680.

EXAMPLE 29 Synthesis ofNH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COAsm(β-NAc(Ac)₃)N[(CH₂)₁₇CH₃]₂

(RPR 131283 A) (45)

a-Fmoc-Asm-β-Glc-NAc(Ac)₃-dioctadecylamine (44)

The product is synthesized by coupling of Fmoc-Asm-β-Glc-NAc(Ac)₃—OH anddioctadecylamine by method F.

TLC Rf=0.67 (CHCl₃/MeOH, 9:1); HPLC, Rt=25.31 min (H₂O/MeCN: 3 min[60/40], 3-20 min [0/100], 35 min [0/100];

b—NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂CO Asm (β-NAc(Ac)₃N[(CH₂)₁₇CH₃]₂ (45)

Cleavage of the Fmoc group from product (45).

20 ml of DMF and 2 ml of diethylamine are poured onto 0.7 g of product(45). After stirring for 6 hours, the medium is concentrated in vacuo.

The product obtained is coupled with product (3) according to method G.The product is purified by semi-preparative HPLC and the fractions areanalysed by analytical HPLC.

HPLC, Rt=15.35 min (H₂O/MeCN: 3 min [60/40], 3-20 min [0/100], 35 min[0/100]; MH⁺: 1207.

EXAMPLE 30 Large-Scale Synthesis in Solution of Product (6)

Product (6) is synthesized by carrying out a reductive alkylation onspermine in the presence of NaCNBH₃ and glyoxylic acid in solution.

18.2 g of spermine, 500 ml of methanol and 2 g of NaCNBH₃ are loadedinto a 2 1 round-bottomed flask. The solution is subjected to vigorousmagnetic stirring. A solution of 8.45 g of glyoxylic acid in 300 ml ofmethanol is run in, via a pressure-equalized dropping funnel, over 100minutes. Stirring is continued overnight. The amines are protected withthe Boc group as follows: 14 ml of TEA are run into the medium, followedby 100 g of di-tert-butyl dicarbonate dissolved in 200 ml of THF.Stirring is continued overnight. The medium is concentrated under vacuumand the product is taken up in ethyl acetate (250 ml), rinsed with KHSO₄(6×100 ml) and then with saturated NaCl solution (3×100 ml), dried overMgSO₄ and evaporated under vacuum. The product is purified on a columnof silica with CHCl₃/MeOH (9:1) as eluent. The fractions containing theproduct are identified by TLC, combined and evaporated under vacuum togive 10 g of product (6) (17% yield for the total synthesis).

The analytical HPLC, mass spectral and NMR analyses are identical tothose of the product obtained by the solid-phase method.

EXAMPLE 31 Influence of the (amines/phosphates) Charge Ratio on theEfficacy of (7) RPR 120534A, (6) RPR 120535A and (9) RPR 120531ATransfection

Samples of 1×10⁵ cells [NIH 3T3, 3LL or SMC rabbit] in exponentialgrowth phase on 2 cm² are treated with lipofectant/pCMV-LUC solutions,having variable charge ratios, for 2 hours at 37° C. under 5% CO₂; eachsample receives 2 μg of nucleic acid. An investigation of the expressionof the reporter gene is carried out after addition of foetal calf serumto a final concentration of 8% followed by incubation for 40 hours in aCO₂ oven.

The luciferase activity is assayed by light emission [RLU=relative lightunit] in the presence of luciferin, coenzyme A and ATP for 10 secondsand is given relative to 2000 treated cells. The results obtained arereported in FIGS. (1), (2) and (3).

From studying these figures, it emerges clearly that the presence of aglycine in the “spacer” arm between the lipid part and the polyaminemakes it possible to obtain a better transfection efficacy for nanomolescationic lipid/low μg DNA ratios.

EXAMPLE 32 Influence of the (amines/phosphates) Charge Ratio on theEfficacy of (20) RPR 120527A, (19) RPR 120528A, (17) RPR 120526A and(16) RPR 120525A Transfection

Samples of 1×10⁵ NIH 3T3 cells in exponential growth phase on 2 cm² aretreated with lipofectant/pCMV-LUC solutions, having variable chargeratios, for 2 hours at 37° C. under 5% CO₂; each sample receives 1 μg ofnucleic acid. An investigation of the expression of the reporter gene iscarried out after addition of foetal calf serum to a final concentrationof 8% followed by incubation for 40 hours in a CO₂ oven.

The luciferase activity is assayed in the supernatant obtained afterlysis of the cells, by light emission [RLU=relative light unit] for 10seconds and is given relative to an mg of protein. The results obtainedare reported in figure (4). The advantage of the presence of the glycineresidue in the “spacer” arm is again demonstrated in this example.

EXAMPLE 33 Influence of the Length of the Spacer Arm on the TransfectionEfficacy (6) RPR 120535, (41) RPR 122786 and (42) RPR 128506

Samples of 1×10⁵ cells [NIH3T3 and HeLa] in exponential growth phase on2 cm² are treated with cationic lipid/pCMV-Luc mixtures, having variableconcentrations of cationic lipid, at 37° C. in a humid atmosphere under5% CO₂, for 2 hours in the absence of serum proteins. The cell growthmedium is then supplemented with foetal calf serum to a finalconcentration of 8% and the transgenic expression is measured after anadditional 40 hours of incubation in a CO2 oven.

The structural characteristics of the spacer arms are as follows:

RPR No. Spacer arm 122786 — 120535 Gly 128506 NH₂(CH₂)₅CO

Table I below gives the results obtained, which are expressed for themaximum efficacies obtained with each of the test products.

TABLE I Cationic lipid HeLa cells NH3T3 cells Experi- RPR120535 (6) 1.2× 10⁶ ± 9.7 × 10⁴ (4) 8.7 × 10⁷ ± ment 1 4.5 × 10⁶ (4) RPR122786 (41)2.3 × 10⁶ ± 1.6 × 10⁵ (8) 4.6 × 10⁷ ± 5.0 × 10⁶ (8) Experi- RPR120535(6) 2.4 × 10⁶ ± 3.2 × 10⁵ (6) 7.2 × 10⁷ ± ment 2 8.3 × 10⁶ (6) RPR128506(42) 1.8 × 10⁶ ± 1.3 × 10⁵ (6) 5.0 × 10⁷ ± 1.5 × 10⁶ (6)

The transfection efficacies are given in RLU/10s/2×103 cells treated.The ratios—nanomoles of lipid/μg of DNA—are indicated in brackets.

Different plasmids were used for Experiments 1 and 2, at a dose of 1 μgand 0.5 μg of DNA/1×105 cells respectively in Experiments 1 and 2.

EXAMPLE 34 Influence of the Structure of the Spacer Arm on theTransfection Efficacy (6) RPR 120535

Under experimental conditions identical to those described in theprevious example, but with the introduction of an additional line (3LLcells), we compared the transfection efficacies obtained with thecationic lipid (6) RPR 120535 modified by substitution on the “spacerarm” with an Arg-type, Lys-type or Glu-type group.

The structures of the various spacer arms are as follows:

RPR No. SPACER ARM 120531 Arg 121650 Arg(Z₂) 127888 Lys 122759

122760

128142

120535 Gly 123027 GluOBz 126097 Glu

The transfection efficacies are given in RLU/10s/2×103 cells treated.

Different plasmids were used for Experiments 1 and 2 at a dose of 0.5 μgand 1 μg of DNA/1×105 cells respectively in Experiments 1 and 2. Fromthe analysis of the results, it emerges that, depending on the cellsconsidered, the presence of a preferably substituted amino acid chaininduces a better transfection efficacy.

TABLE II NIH3T3 lipid HeLa cells cells 3LL cells Experi- RPR120535 1.0 ×10⁶ ± 6.5 × 10⁷ ± ment 1 1.9 × 10⁵ 4.8 × 10⁶ RPR120531 3.7 × 10⁵ ± 1.6 ×10⁷ ± 1.0 × 10⁵ 2.0 × 10⁶ RPR121650 2.6 × 10⁶ ± 9.1 × 10⁷ ± 1.8 × 10⁵2.7 × 10⁷ Experi- RPR120535 2.2 × 10⁶ ± 1.7 × 10⁶ ± ment 2 3.3 × 10⁵ 1.0× 10⁵ RPR127888 3.1 × 10⁵ ± 1.4 × 10⁵ ± 2.9 × 10⁴ 1.7 × 10⁴ RPR1227601.4 × 10⁶ ± 2.2 × 10⁵ RPR122759 7.7 × 10⁵ ± 3.7 × 10⁵ ± 1.1 × 10⁵ 5.4 ×10⁴ RPR128142 6.3 × 10⁶ ± 9.1 × 10⁴ ± 6.3 × 10⁵ 9.3 × 10³ RPR126097 3.6× 10⁶ ± 3.0 × 10⁵ ± 3.6 × 10⁵ 2.3 × 10⁴ RPR123027 1.0 × 10⁶ ± 6.9 × 10⁵± 4.1 × 10⁴ 1.1 × 10⁵

EXAMPLE 34 Influence of the Presence of DOPE in the Lipofectant/DNAMixture (9) RPR 120531A

According to the same procedure as that used in Example 31, DOPE(dioleoylphosphatidylethanolamine) is added to the cationic lipid (9)RPR 120531A in variable molar ratios, before adding DNA to thetransfection mixture.

The luciferase activity is assayed in the supernatant after lysis of thecells and is given relative to an mg of protein (FIG. 5).

The presence of DOPE in the lipofectant mixtures makes it possible toimprove the transfection efficacy when the concentration of (9) RPR120531A is low.

EXAMPLE 35 Effect of Serum on the Efficacy of (6) RPR 120535A, (9) RPR120531A and (10) RPR 121650A Transfection

Samples of 1×10⁵ cells [NIH 3T3 or Hela] in exponential growth phase on2 cm² are treated with lipofectant/pCMV-LUC solutions (3 nanomollipofectant/μg DNA) in the absence of serum for 2 hours or in thepresence of serum in the culture medium. In this example, each samplereceives 2 μg of DNA. The expression of luciferase is studied in thesupernatant of the cell lysates, expressed as RLU/10s and given relativeto an mg of protein. From the analysis of the results, it emerges thatthe presence of serum has no appreciable effect on the transfection.

EXAMPLE 36 Influence of the Nucleic Acid Concentration in theDNA/lipofectant Mixtures (20) RPR 120527A, (19) RPR 120528A, (17) RPR120526A and (16) RPR 120525A

Under the conditions described in Example 31, samples of NIH 3T3 cellsare transfected under conditions in which the nanomoles lipofectant/μgof DNA ratios are optimized—see Example 30—[ratio=6 for (20) RPR 120527Aand (19) RPR 120528A—ratio=3 for (17) RPR 120526A and (16) RPR 120525A].The amounts of DNA provided to each sample range from 0.5 to 2 μg. Theresults are reported in FIG. 6.

The transfection of 1×10⁵ cells in exponential growth phase with 1 μg ofplasmid DNA appears to be a good choice; indeed, the increase in theamount of DNA used leads to an increase in the concentration of cationiclipid in contact with the cells and thus to toxicity problems in certaincases. At a lower DNA concentration, the proportionality with thetransfection efficacy is no longer obtained.

EXAMPLE 37 Tests of in Vivo Transfection with Lipopolyamines Accordingto the Invention.

A solution containing a lipopolyamine according to the invention,prepared as described above, is injected into a tumour 7 days afterimplantation, the mouse being anaesthetized with a ketamine+xylazinemixture. Two days after the injection, tumour tissues are removed,weighed and then chopped up and ground in 500 μl of lysis buffer(Promega Cell Lysis Buffer E153 A). After centrifugation (20,000 g for10 minutes), 10 μl are taken out and used to evaluate the luciferaseactivity by measuring the total light emission obtained after mixingwith 50 μl of reagent (Promega Luciferase Assay Substrate) in a Lumat LB9501 luminometer (Berthold), with integration over 10 seconds. Theresulting activity is expressed as RLUs (Relative Light Units) estimatedfor the entire tumour lysis supernatant, or as RLUs per μg of DNAinjected. Table III gives the results obtained.

TABLE III Plasmid Peptide Cationic lipid Result, RLU/tumour [DNA]pept/DNA nmol/μg standard Reference μg/tumour μg/μl reference w/wreference DNA mean deviation n pXL2622 10 0.5 (KTPKKAKKP)₂ 1.5  (9) 3679 258 414 286 9 (SEQ ID NO.1) pXL2622 10 0.5 (KTPKKAKKP)₂ ″  (6) ″ 395433 219 333 10  pXL2622 10 0.5 ″ ″ (16) ″  67 994  82 527 8 pXL2622 100.5 ″ ″ (19) ″  59 209  54 375 9

2 1 9 PRT Artificial Sequence Description of Artificial SequenceCOMPLETELY SYNTHESIZED 1 Lys Thr Pro Lys Lys Ala Lys Lys Pro 1 5 2 8 PRTArtificial Sequence Description of Artificial Sequence COMPLETELYSYNTHESIZED 2 Ala Thr Pro Ala Lys Lys Ala Ala 1 5

What is claimed is:
 1. A lipopolyamine in D, L or L,D form or a saltthereof, of the general formula I

in which: R1, R2 and R3 represent, independently of each other, ahydrogen atom or a group —(CH₂)q—NRR′ with q able to range between 1, 2,3, 4, 5 and 6, and doing so independently between the various groups R1,R2 and R3 and R and R′ representing, independently of each other, ahydrogen atom or a group —(CH₂)q′-NH2, q′ being able to range between 1,2, 3, 4, 5 and 6, and doing so independently between the various groupsR and R′, m and p represent, independently of each other, an integerwhich may vary between 1 and 6, and n represents an integer which mayvary between 0 and 6, wherein, when n is equal to 0, then at least oneof R1 and R2 is other than hydrogen, and when n is greater than 1, m isable to take different values and R3 is able to take different meaningswithin the general formula I, and R4 represents a group of generalformula II

in which: R6 and R7 represent, independently of each other, a hydrogenatom or a saturated or unsaturated C10 to C22 aliphatic radical with atleast one of the two groups being other than hydrogen, u is an integerchosen between 0 and 10 with, when u is an integer greater than 1, R5,X, Y and r able to have different meanings within the different units(X-(CHR5)r-Y) X represents an oxygen or sulphur atom or an amine group,Y represents a carbonyl group or a methylene group R5 represents ahydrogen atom or a side chain of a natural amino acid, which issubstituted if necessary, and r represents an integer ranging between 1and 10 with, when r is equal to 1, R5 representing a side chain of asubstituted or unsubstituted natural amino acid and, when r is greaterthan 1, R5 representing a hydrogen atom.
 2. The lipopolyamine accordingto claim 1, selected from the group consisting of the following formulae

in which R4, R6 and R7 are defined as in claim
 1. 3. A lipopolyamineaccording to claim 2, characterized in that R4 represents therein anNR6R7 group with R6 and R7 appearing in subformulae III to XII as anidentical group selected from the group consisting of (CH₂)₁₇CH₃,(CH₂)₁₁CH₃, (CH₂)₁₃CH₃ and (CH₂)₁₂CH₃.
 4. A lipopolyamine according toclaim 1, characterized in that it is combined with an extra- orintracellular targeting element.
 5. A lipopolyamine according to claim4, characterized in that it incorporates the targeting element in theamino acid side chain featured by substituent R5.
 6. A lipopolyamineaccording to claim 4, characterized in that it is a ligand of a cellreceptor present at a surface of a target cell.
 7. A lipopolyamineaccording to claim 4, characterized in that the targeting element isrepresented by a nuclear localization signal sequence.
 8. Alipopolyamine according to claim 1, characterized in that it it isselected from the group consisting of:H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlyN[(CH₂)₁₇—CH₃]₂H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂CON[(CH₂)₁₇—CH₃]₂H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COArgN[(CH₂)₁₇—CH₃]₂H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COArg(Z)₂N[(CH₂)₁₇—CH₃]₂H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLys(rhodamine)N[(CH₂)₁₇—CH₃]₂H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLys(biotinyl)N[(CH₂)₁₇—CH₃]₂{H₂N(CH₂)₃}₂N(CH₂)₄N{(CH₂)₃NH₂}(CH₂)₃NHCH₂COGlyN[(CH₂)₁₇—CH₃]₂{H₂N(CH₂)₃}₂N(CH₂)₄N{(CH₂)₃NH₂}(CH₂)₃NHCH₂CON[(CH₂)₁₇—CH₃]₂{H₂N(CH₂)₂}₂N(CH₂)₂NHCH₂COGlyN[(CH₂)₁₇—CH₃]₂{H₂N(CH₂)₂}₂N(CH₂)₂NHCH₂CON[(CH₂)₁₇—CH₃]₂H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlyN[(CH₂)₁₇]₂H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂CON[(CH₂)₁₇]₂H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COArgN[(CH₂)₁₇]₂H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COArg(Z)₂N[(CH₂)₁₇—CH₃]₂H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLys(rhodamine)N[(CH₂)₁₇—CH₃]₂H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLys(biotinyl)N[(CH₂)₁₇—CH₃]₂{H₂N(CH₂)₃}₂N(CH₂)₄N{(CH₂)₃NH₂}(CH₂)₃NHCH₂COGlyN[(CH₂)₁₇—CH₃]₂{H₂N(CH₂)₃}₂N(CH₂)₄N{(CH₂)₃NH₂}(CH₂)₃NHCH₂CON[(CH₂)₁₇—CH₃]₂{H₂N(CH₂)₂}₂N(CH₂)₂NHCH₂COGlyN[(CH₂)₁₇—CH₃]₂{H₂N(CH₂)₂}₂N(CH₂)₂NHCH₂CON[(CH₂)₁₇—CH₃]₂NH₂(CH₂)₃NH(CH₂)₄N[(CH₂)₃NH₂]CH₂COGlyN[(CH₂)₁₇CH₃]₂NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLysN[(CH₂)₁₇CH₃]₂NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLys [Cl—Z]N[(CH₂)₁₇CH₃]₂NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLys [CHO]N[(CH₂)₁₇CH₃]₂NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLys [Cholesteryl]N[(CH₂)₁₇CH₃]₂NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COLys [Arachidonyl]N[(CH₂)₁₇CH₃]₂NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGluN[(CH₂)₁₇CH₃]₂NH₂(CH₂)₃NH(C₂)₄NH(CH₂)₃NHCH₂COGlu [N(CH₃)₂]N[(CH₂)₁₇CH₃]₂NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlu [O—Bz]N[(CH₂)₁₇CH₃]₂NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlu [Galactosamide]N[(CH₂)₁₇CH₃]₂NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlu [Glucosamide]N[(CH₂)₁₇CH₃]₂NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlu [Mannosamide]N[(CH₂)₁₇CH₃]₂NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH(CH₂)₃CON[(CH₂)₁₇CH₃]₂NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂CONH(CH₂)₅CON[(CH₂)₁₇CH₃]₂NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlyN[(CH₂)₁₁CH₃]₂NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlyN[(CH₂)₁₁CH₃]₂NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlyN[(CH₂)₁₂CH₃]₂ andNH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlyN[(CH₂)₁₃CH₃]₂.
 9. A pharmaceuticalcomposition, comprising at least one lipopolyamine according to claim 1and at least one nucleic acid.
 10. The composition according to claim 9,characterized in that the nucleic acid is a deoxyribonucleic acid. 11.The composition according to claim 9, characterized in that the nucleicacid is a ribonucleic acid.
 12. The composition according to claim 9,characterized in that the nucleic acid is chemically modified.
 13. Thecomposition according to claim 9, characterized in that the nucleic acidis an antisense sequence.
 14. The composition according to claim 9,characterized in that the nucleic acid contains a therapeutic gene. 15.A pharmaceutical composition comprising a nucleic acid, a lipopolyamineaccording to claim 1 and an adjuvant, wherein the lipopolyamine andnucleic acid associate to form a lipopolyamine/nucleic acid complex andthe adjuvant combines with the lipopolyamine/nucleic acid complex.
 16. Acomposition according to claim 15, characterized in that the adjuvant isone or more neutral lipids.
 17. A composition according to claim 16,characterized in that the neutral lipid or lipids are selected from thegroup consisting of natural zwitterionic lipids, synthetic zwitterioniclipids, and lipids lacking any ionic charge under physiologicalconditions.
 18. A composition according to claim 17, characterized inthat the neutral lipid or lipids are lipids with 2 fatty chains.
 19. Acomposition according to claim 15, characterized in that it comprisesfrom 0.01 to 20 equivalents of adjuvant per one equivalent of nucleicacid on a weight/weight basis.
 20. A composition according to claim 9,characterized in that it comprises a vehicle which is pharmaceuticallyacceptable for an injectable formulation.
 21. A composition according toclaim 9, characterized in that it comprises a vehicle which ispharmaceutically acceptable for an application to the skin and/or themucous membranes.
 22. A process for the preparation of a lipopolyamineaccording to claim 1, comprising the step of coupling at least one lipidmoiety with at least one asymmetric polyamine moiety, wherein theasymmetric polyamine moiety was obtained by bimolecular reaction betweenan alkylating agent covalently attached to a solid support and asymmetric polyamine.
 23. The process according to claim 22,characterized in that the coupling of the lipid moiety with theasymmetric polyamine moiety is carried out on the solid support to whichthe asymmetric polyamine moiety is bound and in that the lipopolyaminethus obtained is recovered.
 24. The process according to claim 23,characterized in that it involves the introduction of labelling agents,sugars or fluorescent probes onto the lipopolyamine.
 25. A method forthe transfection of cells comprising contacting a cell with thecomposition of claim
 9. 26. The lipopolyamine according to claim 4,wherein the targeting element is selected from the group consisting ofsugars, peptides, oligonucleotides, steroids, and lipids.
 27. Thelipopolyamine of claim 26 wherein the targeting element is selected fromthe group consisting of antibodies, antibody fragments, cell receptorligands, fragments of cell receptor ligands, receptors, and receptorfragments.
 28. The lipopolyamine of claim 27 wherein the targetingelement is selected from the group consisting of ligands for growthfactor receptors, ligands for cytokine receptors, ligands for celllectin receptors, ligands for receptors for adhesion proteins, integrinreceptors, transferrin receptors, HDL lipid receptors, and LDL lipidreceptors.
 29. The lipopolyamine of claim 1 further comprising alabelling agent selected from the group consisting of biotin, rhodamine,folate, folate derivatives, linear peptides, cyclic peptides, andpseudopeptide sequences containing the Arg-Gly-Asp epitope.
 30. Apharmaceutical composition for the transfection of nucleic acidcomprising a nucleic acid, a lipopolyamine according to claim 1 and anadjuvant, wherein the presence of the adjuvant increases thetransfection power over the transfection power in the absence ofadjuvant.
 31. The composition of claim 17, wherein the neutral lipid orlipids are selected from the group consisting ofdioleoylphosphatidylethanolamine (DOPE);oleoylpalmitoylphosphatidylethanolamine (POPE); distearoyl, -palmitoyl,-myristoyl phosphatidyl-ethanolamine; phosphatidylglycerols;diacylglycerols; glycosyldiacylglycerols; cerebrosides; sphingolipids;and asialogangliosides.
 32. The composition of claim 30, wherein theadjuvant is a compound involved in the condensation of the nucleic acid.33. A composition according to claim 32, characterized in that the saidcompound is derived partly or totally from a histone, from a nucleolineand/or from a protamine.
 34. A composition according to claim 32,characterized in that the compound comprises peptide units (KTPKKAKKP)and/or (ATPAKKAA) repeated continuously or non-continuously, and whereinthe number of peptide units ranges between 2 and 10.